Analysis of the Indacaterol-Regulated Transcriptome in Human Airway

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1521-0103/366/1/220–236$35.00 https://doi.org/10.1124/jpet.118.249292 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 366:220–236, July 2018 Copyright ª 2018 by The American Society for Pharmacology and Experimental Therapeutics

Analysis of the Indacaterol-Regulated Transcriptome in Human Airway Epithelial Cells Implicates Gene Expression Changes in the s Adverse and Therapeutic Effects of b2-Adrenoceptor Agonists

Dong Yan, Omar Hamed, Taruna Joshi,1 Mahmoud M. Mostafa, Kyla C. Jamieson, Radhika Joshi, Robert Newton, and Mark A. Giembycz Departments of Physiology and Pharmacology (D.Y., O.H., T.J., K.C.J., R.J., M.A.G.) and Cell Biology and Anatomy (M.M.M., R.N.), Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada Received March 22, 2018; accepted April 11, 2018 Downloaded from

ABSTRACT The contribution of gene expression changes to the adverse and activity, and positive regulation of neutrophil chemotaxis. The therapeutic effects of b2-adrenoceptor agonists in asthma was general enriched GO term extracellular space was also associ- investigated using human airway epithelial cells as a therapeu- ated with indacaterol-induced genes, and many of those, in- tically relevant target. Operational model-fitting established that cluding CRISPLD2, DMBT1, GAS1, and SOCS3, have putative jpet.aspetjournals.org the long-acting b2-adrenoceptor agonists (LABA) indacaterol, anti-inflammatory, antibacterial, and/or antiviral activity. Numer- salmeterol, formoterol, and picumeterol were full agonists on ous indacaterol-regulated genes were also induced or repressed BEAS-2B cells transfected with a cAMP-response element in BEAS-2B cells and human primary bronchial epithelial cells by reporter but differed in efficacy (indacaterol $ formoterol . the low efficacy LABA salmeterol, indicating that this genomic salmeterol $ picumeterol). The transcriptomic signature of effect was neither unique to indacaterol nor restricted to the indacaterol in BEAS-2B cells identified 180, 368, 252, and BEAS-2B airway epithelial cell line. Collectively, these data 10 genes that were differentially expressed (.1.5- to ,0.67- suggest that the consequences of inhaling a b2-adrenoceptor fold) after 1-, 2-, 6-, and 18-hour of exposure, respectively. Many agonist may be complex and involve widespread changes in at ASPET Journals on September 30, 2021 upregulated genes (e.g., AREG, BDNF, CCL20, CXCL2, EDN1, gene expression. We propose that this genomic effect repre- IL6, IL15, IL20) encode proteins with proinflammatory activity sents a generally unappreciated mechanism that may contribute and are annotated by several, enriched gene ontology (GO) to the adverse and therapeutic actions of b2-adrenoceptor terms, including cellular response to interleukin-1, cytokine agonists in asthma.

Introduction recently, the cAMP-dependent phosphorylation of substrates that directly inhibit myosin from generating force on actin b Inhaled 2-adrenoceptor agonists are a mainstay asthma filaments was believed to explain the improvement in lung therapy, providing symptomatic relief by effecting rapid and function (Giembycz and Raeburn, 1991). However, recent evi- prolonged airway smooth muscle (ASM) relaxation. Until dence suggests that b2-adrenoceptor agonists can maintain airway caliber by genomic mechanisms. Studies by Holden b This study was supported by a project grant from the Canadian Institutes et al. (2011) revealed that in ASM, long-acting 2-adrenoceptor for Health Research (PJT 152904), The Lung Association, Alberta & NWT, and agonists (LABAs) induced the gene encoding regulator of an unrestricted research grant from Gilead Sciences Inc., Seattle, WA. K.C.J., G protein signaling 2 (RGS2). RGS2 is a GTPase-activating M.M.M., O.H., and T.J. are recipients of studentships awarded by The Lung Association, Alberta & NWT. D.Y. is supported by Alberta Innovates. K.C.J. is protein that terminates signaling mediated by G protein- a recipient of a University of Calgary Eyes High Doctoral Recruitment coupled receptors (GPCRs) that act through Gq (Heximer, Scholarship. Real-time PCR was facilitated by an equipment and infrastruc- 2004). As many spasmogens (e.g., acetylcholine, histamine, ture grant from the Canadian Fund of Innovation and the Alberta Science and Research Authority. The authors state no conflict of interest. and leukotrienes) act in this way, the expression of RGS2 in 1Current affiliation: Global Development Operations, Novartis Healthcare ASM may represent a novel mechanism that protects against Pvt. Ltd, Salarpuria-Sattva Knowledge City, Raidurg, Hyderabad, India. https://doi.org/10.1124/jpet.118.249292. bronchoconstriction and improves lung function in asthma s This article has supplemental material available at jpet.aspetjournals.org. (Holden et al., 2011). It follows, therefore, that b2-adrenoceptor

ABBREVIATIONS: ANOVA, analysis of variance; ASM, airway smooth muscle; b2A, 8-hydroxy-5-((R)-1-hydroxy-2-methylaminoethyl)-1H-quinolin- 2-one; CMV, cytomegalovirus; CRE, cAMP response element; CREB, cAMP response element–binding protein; DAVID, database for visualization and integrated discovery; DCITC, (5(2-(((19-(49-isothiocyanatephenylamino)thiocarbonyl)amino)-2-methylpropyl)amino-2-hydroxypropoxy)-3,4- dihydrocarbostyril; DEGs, differentially expressed genes; FDR, false discovery rate; FZR, frizzled receptor; GFP, green fluorescent protein; GINA, global initiative for asthma; GO, gene ontology; HBECs, human bronchial epithelial cells; ICS, inhaled corticosteroid; KEGG, Kyoto encyclopedia of genes and genomes; LABA, long-acting b2-adrenoceptor agonist; MAP, mitogen-activated protein; MOI, multiplicity of infection; PCR, polymerase chain reaction; PKA, cAMP-dependent protein kinase; PKI, protein kinase inhibitor; RGS2, regulator of protein signaling 2; SABA, short-acting b2- adrenoceptor agonist; SFM, serum-free medium.

220 Indacaterol-Regulated Transcriptome in Human Airway Epithelia 221 agonists may upregulate other genes in the lungs that can also model-fitting (Black and Leff, 1983). The transcriptomic signa- impart therapeutic benefit (Moodley et al., 2013; Giembycz and ture of the LABA indacaterol was also determined in BEAS- Newton, 2015). 2B cells by microarray and analyzed bioinformatically, with key Although b2-adrenoceptor agonists are unquestionably ben- gene expression changes being confirmed in human primary eficial in asthma, chronic use as a monotherapy is often bronchial epithelial cells (HBECs). associated with a loss of disease control and an increased risk of asthma-related mortality (Pearce et al., 1991; Cockcroft and Sears, 2013). This problem first arose in England and Wales in Materials and Methods the 1960s (Speizer et al., 1968a) and recurred in New Zealand Generation of a Stable CRE Luciferase Reporter in BEAS- a decade later (Jackson et al., 1982). Epidemiologic studies 2B Cells. Cells were transfected with 8 mg of plasmid DNA (pADneo2- indicate that the rise in asthma mortality correlates with the C6-BGL) using Lipofectamine 2000 (Invitrogen/Thermo Fisher increased use of high-dose, nonselective compounds, such as Scientific, Burlington, ON, Canada) to generate 6CRE BEAS-2B cells isoprenaline (Speizer et al., 1968b; Stolley, 1972; Stolley and as described previously (Meja et al., 2004). Submersion Culture of BEAS-2B Cells. Native and 6CRE Schinnar, 1978), and short-acting b2-adrenoceptor agonists BEAS-2B reporter cells were cultured for 3 days under a 5% CO /air (SABAs), such as fenoterol (Crane et al., 1989). At the time, b1- 2 adrenoceptor-mediated cardiotoxicity was thought to be a atmosphere at 37°C in 24-well plastic plates (Corning Life Sciences, Lowell, MA) in keratinocyte-containing, serum-free medium (SFM) causal factor (Speizer et al., 1968b). The finding that iso-

supplemented with epidermal growth factor (5 ng/ml), bovine pituitary Downloaded from prenaline and fenoterol have high intrinsic activity at cardiac extract (50 mg/ml), penicillin (100 mg/ml), and streptomycin (100 IU/ml), b 1-adrenoceptors (Wilson and Lincoln, 1984; Steinfath et al., and for a further 24 hours in SFM (Greer et al., 2013). At this time, 1992) that could be activated secondarily to systemic exposure cultures were confluent and were used to assess reporter activation and is consistent with that interpretation. Weak selectivity of gene expression. fenoterol for the b2-adrenoceptor over the b1-subtype (Baker Infection of BEAS-2B Cells with an Adenovirus Encoding an et al., 2015) also supports that idea. However, the discovery Endogenous Inhibitor of cAMP-Dependent Protein Kinase. that the highly selective LABA salmeterol (Baker et al., 2015) Subconfluent (∼70%), BEAS-2B cells were infected [multiplicity of jpet.aspetjournals.org is also associated with a loss of asthma control and an increase infection (MOI) 5 25] with a replication-deficient adenovirus vector a in mortality in a subset of patients with asthma (Nelson et al., (Ad5.CMV.PKI ) containing a 251-bp DNA fragment encoding the complete amino acid sequence of the a-isoform of cAMP-dependent 2006) casts doubt on cardiovascular toxicity being the major a b protein kinase (PKA) inhibitor (PKI ) positioned downstream of the cause. Instead, it seems probable that overuse of 2-adreno- constitutively active cytomegalovirus (CMV) immediate early promoter ceptor agonists, as a class, masks an underlying inflammation (Meja et al., 2004). The same vector encoding green fluorescent protein (McIvor et al., 1998) that leads to an increase in disease (GFP) was used as a control. After 48 hours, cells were exposed to severity and associated adverse events (Pearce et al., 1991; salmeterol (100 nM; 2 hours) and processed for gene expression by real- at ASPET Journals on September 30, 2021 Cockcroft and Sears, 2013). Accordingly, the Global Initiative time polymerase chain reaction (PCR) (vide infra). for Asthma (GINA) treatment guidelines advocate that SABAs Treatment of BEAS-2B Cells. Native and 6CRE BEAS-2B b should be used on an as-needed basis for symptoms relief (www. reporter cells were treated with the 2-adrenoceptor agonists of ginasthma.org). GINA also recommends that a LABA should interest. Reporter cells were lysed at 6 hours in 1 firefly luciferase always be administered concurrently with an inhaled corti- lysis buffer (Biotium, Hayward, CA), and luciferase activity was measured by luminometry. Data are expressed as fold increase in costeroid (ICS), which has been shown to protect against luciferase activity using time-matched activity values in vehicle- salmeterol-induced toxicity (Cates et al., 2014). This latter treated cells as the denominator. RNA was extracted from native finding is informative because ICSs suppress the immune cells and subjected to real-time PCR (vide infra). system, implying that the etiology of SABA- and LABA- Submersion Culture of Primary Cells. HBECs were prepared induced adverse effects has an inflammatory basis. by proteinase digestion of nontransplanted, normal airways harvested Collectively, these data suggest that the consequences of from five healthy, nonasthmatic donors obtained from the Interna- inhaling a b2-adrenoceptor agonist are more complex than tional Institute for the Advancement of Medicine (Edison, NJ; see originally thought and may involve widespread changes in Supplemental Table 1 for demographics). Cells were seeded in 12-well gene expression. Establishing a genomic effect has additional plates (Corning) containing bronchial epithelial cell growth medium m clinical implications, because it implies that the pulmonary (Lonza, Walkersville, MD) supplemented with penicillin (50 g/ml) and streptomycin (10 mg/ml), and maintained under a 5% CO /air atmo- actions of these drugs in asthma are not restricted to ASM. 2 sphere at 37°C until ∼80% confluent (∼14 days; medium changed every Indeed, many structural elements within the lungs, such as 2–3 days). HBECs were cultured for a further 24 hours in supplement- b the airway epithelium, express a high density of 2-adreno- free, basal medium (Lonza) and processed for real-time PCR (vide infra). ceptors that are efficiently coupled to adenylyl cyclase (Penn Ethics approval for the use of human tissues has been granted by the et al., 1994) and could represent a therapeutically relevant Conjoint Health Research Ethics Board of the University of Calgary. target. Microarray and Data Processing. BEAS-2B cells in In this study, we assessed whether gene expression changes keratinocyte-containing SFM without supplements were cultured for in human bronchial epithelial cells could contribute to the 1, 2, 6, and 18 hours with indacaterol (10 nM) or vehicle (N 5 4 at each time-point). Total RNA was extracted (vide infra), quantified (Nano- adverse effects of b2-adrenoceptor agonists in asthma. The prospect that differentially expressed genes (DEGs) could Drop 2000; Thermo Fisher Scientific), and the quality of each sample was assessed using the Agilent 2100 Laboratory-on-a-Chip System impart therapeutic benefit was also explored. To address these b (Santa Clara, CA) before being processed for gene profiling (Expression possibilities, the pharmacodynamics of four 2-adrenoceptor Analysis Inc., Durham, NC). Each RNA sample was converted into a agonists (indacaterol, salmeterol, formoterol, and picumeterol) biotinylated cRNA target using a 39-IVT Expression Kit (Affymetrix/ that define the activation of a cAMP-response element (CRE) Thermo Fisher Scientific) and hybridized to an Affymetrix GeneChip luciferase reporter transfected into the BEAS-2B human Human Genome U133 Plus 2.0 Array, which provides an analysis of airway epithelial cell line were compared using operational ∼47,400 transcripts from 54,613 probe sets. Chips were stained, 222 Yan et al. washed, and the fluorescence intensity was measured. The microarray pharmacological effect (E) and agonist concentration (Black and Leff, images were normalized using the probe logarithmic intensity error 1983). Algebraically, (PLIER) algorithm and stored as .chp files. Signals from the four tn×½ n replicates for each probe set were averaged and the relative expression 5 Em× A E n n n (2) patterning was implemented in Transcriptome Analysis Console (TAC ðKA 1 ½AÞ 1 t ×½A v4.0; Affymetrix). Data were analyzed at each time-point and visualized by generating volcano plots and heat maps. The P statistic was adjusted where Em is the theoretical maximum response of the tissue, KA is the using the Benjamini and Hochberg false discovery rate (FDR), with agonist equilibrium dissociation constant, n is the slope of the step-up, procedure, and significance was set at a value of ,0.1. These relationship between the concentration of agonist-receptor complexes t microarray data have been uploaded to NCBI’s Gene Expression and response, and is the operational efficacy of the agonist, which is Omnibus and are available through accession code GSE106710. the reciprocal of the percentage of agonist-bound receptors required to t Unless indicated otherwise, genes, mRNAs, and proteins are re- give half maximal response. In these analyses, only was allowed to ferred to by the official gene symbol supplied by The National vary between individual E/[A] curves; for all other parameters (i.e., Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). Em, KA and n) a common value was assumed (Black and Leff, 1983; Functional classification of indacaterol-regulated genes (by probe Leff et al., 1990). set) including associated gene ontology (GO) terms was performed Determination of Receptor Reserve. Receptor occupancy- b with the database for visualization and integrated discovery response curves were constructed using KA values determined by 2- (DAVID) bioinformatics resources (v6.8) at medium stringency adrenoceptor depletion. At each concentration of agonist, fractional receptor occupancy was determined, assuming the binding of ligand to

(default settings). For all analyses an enrichment score cut-off Downloaded from b value of .1.3 was applied. This is a relative probability that the 2-adrenoceptor was a noncooperative process according to eq. 3, represents the geometric mean of all enrichment P values of each where RA and Rt represent the number of agonist-occupied receptors annotation term in a given cluster. It is presented on a linear scale and total number of receptors, respectively. whereavalueof1.3isequaltothe2log of 0.05 (Huang et al., 10 R ∕ R 5 ½A ∕ ðK 1 ½AÞ (3) 2009). Pseudogenes, hypothetical genes, noncoding RNAs and A t A uncharacterized sequences lacking annotation were excluded. Drugs and Analytical Reagents. b2A (8-hydroxy-5-((R)-1-hy- Results are reported using enriched GO terms that describe biologic droxy-2-methylamino-ethyl)-1H-quinolin-2-one) and indacaterol were jpet.aspetjournals.org process (GOTERM_BP_DIRECT). When this descriptor was absent synthesized by Gilead Sciences (Seattle, WA). Salmeterol was from from a given gene cluster, molecular function (GOTERM_MF_DIR- GlaxoSmithKline (Stevenage, UK), and formoterol and picumeterol ECT), cellular component (GOTERM_CC_DIRECT), UniProt Se- were provided by AstraZeneca (Mölndal, Sweden). DCITC (5(2-(((19- quence Feature, InterPro, and/or Kyoto Encyclopedia of Genes and (49-isothiocyanatephenylamino)thiocarbonyl) amino)-2-methylpropyl)- Genomes (KEGG) pathway were used. amino-2-hydroxypropoxy)-3,4-dihydrocarbostyril) was a generous gift RNA Isolation, Reverse Transcription, and Real-Time PCR. from Dr. Stephen Baker (University of Florida, Gainesville, FL). All Total RNA was extracted from epithelial cells (RNeasy Mini Kit; drugs were dissolved in dimethyl sulfoxide (DMSO) and diluted to the Qiagen, Mississauga, ON, Canada) and reverse-transcribed using a required working concentrations in SFM. The highest concentration of at ASPET Journals on September 30, 2021 qScript cDNA synthesis kit according to the manufacturer’s instruc- DMSO used in these experiments (0.2% v/v) did not affect any output tions (Quanta Biosciences, Gaithersburg, MD). Real-time PCR anal- measured. ysis of cDNA was performed using the primers shown in Supplemental Statistics. Data points, bars, and values in the text and figure Table 2. Reactions (10 ml) were performed using an ABI StepOnePlus legends represent the mean 6 S.E.M. of N independent determina- instrument (Applied Biosystems/Thermo Fisher Scientific, Foster tions. CRE reporter and gene expression data were analyzed by City, CA) on 2.5 ml of cDNA using Fast SYBR Green chemistry Student’s two-tailed t test or repeated measures, one-way analysis of (Invitrogen/Thermo Fisher Scientific), according to the manufac- variance (ANOVA) as indicated. If the ANOVA F-test P value turer’s guidelines. Gene expression levels were determined from a was ,0.05, differences between treatment groups were analyzed by cDNA standard curve (analyzed simultaneously with test samples) Tukey’s multiple comparison test. Rank order correlations were and are presented as a ratio to GAPDH. Amplification conditions were performed using the method of Pearson. The null hypothesis was as follows: 95°C, 20 seconds; followed by 40 cycles of 95°C, 3 seconds, rejected when P , 0.05. and 60°C, 30 seconds. Dissociation curves (95°C, 15 seconds; 60°C, 1 minute; 95°C, 15 seconds) were constructed to confirm primer specificity. Results Curve-Fitting. Monophasic E/[A] curves were fit by least-squares, Effect of LABAs on CRE-Dependent Reporter Acti- nonlinear, iterative regression to the following form of the Hill vation. On 6CRE BEAS-2B reporter cells, indacaterol, equation (eq. 1; Prism 6; GraphPad Software Inc., La Jolla, CA): formoterol, salmeterol, and picumeterol increased luciferase 2 activity in a concentration-dependent manner. Relative to the 5 1 Emax Emin E Emin n (1) b ðp½A 2 p½AÞ reference compound, 2A, which is a well-recognized pharma- 1 1 10 50 cophore that confers b2-adrenoceptor agonism (Yoshizaki et al., where E is the effect, Emin and Emax are the basal response and 1976), all LABAs were full agonists (intrinsic activity [a]values maximum response, respectively, p[A] is the negative log molar of ∼1) with the following rank order of potency: formoterol . b concentration of the 2-adrenoceptor agonist of interest, p[A]50 is a picumeterol . salmeterol . indacaterol (Fig. 1; Table 1). location parameter equal to the negative log molar concentration of Pharmacodynamics of b -Adrenoceptor-Mediated, 2 2 agonist producing (Emax Emin)/2 and n is the gradient of the E/[A] CRE-Dependent Reporter Activation. Operational model- curve at the p[A] level. 50 fitting of E/[A] curve data before and after treatment of 6CRE Determination of Agonist Equilibrium Dissociation Con- BEAS-2B reporter cells with the alkylating agent DCITC de- stants. Agonist affinities were estimated by fractional, irreversible, termined that the KA values of the four LABAs differed by a b2-adrenoceptor inactivation. E/[A] curves were generated in cells that had been pretreated (60 minutes) with vehicle or the alkylating factor of 182-fold, with picumeterol and indacaterol having the b agent DCITC (100 nM; Deyrup et al., 1998), and washed in SFM. Each highest and lowest affinity for 2-adrenoceptor-mediated repair of E/[A] curves was fit simultaneously to the operational model of porter activation, respectively (Fig. 2, A–D; Table 2). Operational agonism (eq. 2), which describes a theoretical relationship between efficacy values also varied; formoterol and indacaterol had Indacaterol-Regulated Transcriptome in Human Airway Epithelia 223

TABLE 1 Effect of LABAs on CRE-dependent reporter activation in BEAS-2B cells Parameters were derived from the data in Fig. 1. b2A was used as a reference agonist and assigned a value of 1.

Treatment N p[A]50 Emax (fold) a b2A 7 8.70 6 0.04 33.3 6 1.9 1 Formoterol 7 10.43 6 0.06 31.8 6 3.5 0.96 Indacaterol 7 8.84 6 0.08 31.9 6 5.6 0.96 Salmeterol 7 9.11 6 0.10 33.0 6 4.0 0.99 Picumeterol 8 9.72 6 0.07 31.3 6 3.4 0.94

(.1.5-fold induction or ,0.67 repression and .3-fold induction or ,0.33-fold repression). In BEAS-2B cells exposed to indacaterol for 1 hour, 180 DEGs were identified that were either . Fig. 1. Effect of LABAs on reporter activation in 6CRE BEAS-2B cells. upregulated (134) or downregulated (46) by a factor of 1.5-fold E/[A] curves were constructed to formoterol, indacaterol, salmeterol, and and ,0.67, respectively (Fig. 3). After 2 hours of exposure, the Downloaded from picumeterol, from which p[A]50 and intrinsic activity values were derived number of DEGs at these same thresholds had increased to using b2A as a reference agonist. The horizontal dashed line represents baseline luciferase activity. Data points represent the mean 6 S.E.M. of 368 (321 induced and 47 repressed), which had declined to a seven or eight independent observations as indicated in Table 1. value of 252 (205 induced and 47 repressed) by 6 hours (Fig. 3). At 18 hours, nine genes were upregulated and one gene was downregulated by indacaterol (Fig. 3). Similar kinetics of gene ∼10-fold higher efficacy (t ∼45) than did salmeterol and induction were found if no threshold (data not shown) picumeterol (Table 2). The affinity and operational efficacy of and .3-fold/,0.33-fold thresholds were set for significant gene jpet.aspetjournals.org b2A were similar to that of indacaterol (Fig. 2E; Table 2). expression changes, but the number of DEGs was markedly Using the KA value of each LABA, the relationship between higher and lower, respectively (Fig. 3). Figure 4 shows the b2-adrenoceptor occupancy and reporter gene activation was expression level of all probe sets as volcano plots at 1, 2, 6 determined to be curvilinear and deviated significantly from and 18 hours after indacaterol, where red and blue circles the line of identity (at which response is a linear function of indicate .1.5-fold induction and ,0.67-fold repression, occupancy; Fig. 2F). Although the proportion of “spare” respectively. receptors declined incrementally with increasing agonist Preliminary GO analyses at each time-point was performed at ASPET Journals on September 30, 2021 concentration, a receptor reserve was evident at most levels using six generic descriptors: 1) transcriptional regulators of response. Thus, for formoterol and indacaterol, a large b2- (red), 2) transporters, ion channels, and membrane receptors adrenoceptor reserve existed for mediating CRE-dependent (orange), 3) metabolic proteins (yellow), 4) general signaling reporter activation, which is consistent with the high KA/[A]50 molecules, including translational regulators (green), 5) other ratios (Table 2). The occupancy-response relationships of sal- functions (blue), and 6) not assigned (purple). The number of meterol and picumeterol were also similar, but the receptor genes within each of these terms expressed as a percentage of reserves at all levels of reporter activation were considerably the total number of significant gene expression changes at lower. Thus, formoterol and indacaterol required approximately each time-point is presented as a pie chart on the left side 0.7%, 2.6%, and 11% b2-adrenoceptor occupancy to produce 20%, (repressed genes) and right side (induced genes) of each 50%, and 80% of the maximal response, respectively. In contrast, volcano plot shown in Fig. 4. considerably greater receptor occupancy (10%, 30%, and 65%, Transcriptional regulators represented ∼26% of all induced respectively) was necessary for salmeterol and picumeterol to genes in BEAS-2B cells exposed to indacaterol for 1 and 2 hours, produce equivalent levels of reporter activation (Fig. 2F). which had declined to 7% and 0% of the total number of genes at Effect of Indacaterol on Global Gene Expression. the 6- and 18-hour time-points, respectively. The number of Microarray-based expression profiling was used to identify genes encoding signaling molecules (11%–18.5%), metabolic DEGs in BEAS-2B cells that may contribute to the adverse proteins (24%–35%), and transporters, ion channels, and and therapeutic effects of b2-adrenoceptor agonists. The membrane receptors (12%–17%) followed similar, but less LABA, indacaterol (10 nM), was selected for this purpose extreme, induction kinetics (Fig. 5; Supplemental Fig. 1A). because it had high operational efficacy in 6CRE BEAS-2B Genes encoding transcriptional regulators, signaling mole- reporter cells (Table 2), which should have maximized the cules and proteins “not assigned” were rapidly, albeit, tran- number of significant gene expression changes detected using siently repressed at 1 and 2 hours in cells treated with this platform. indacaterol. The expression of genes in the other three groups Immediate and/or delayed targets of cAMP signaling could followed similar, but slower, kinetics with maximum repres- contribute to the activity of b2-adrenoceptor agonists. Accord- sion occurring at 6 hours before returning to pre-stimulation ingly, DEGs were identified at 1, 2, 6, and 18 hours after levels at 18 hours (Fig. 6; Supplemental Fig. 1B). exposure to indacaterol. Of the ∼47,400 probe sets that are All DEGs are listed in Supplemental Tables 3 and 4 and are present on each gene chip, 458, 881, 1408, and 13 had assigned a color (right side column) that corresponds to one of expression-level changes that were statistically different the six generic GO terms listed above. (FDR P , 0.1) from time-matched, vehicle-treated cells at Validation of Gene Expression Changes. Supplemental 1, 2, 6, and 18 hours, respectively. The kinetics of global Figs. 2 and 3 compare, as heat maps, the expression pattern of gene expression changes are shown in Fig. 3 at two thresholds indacaterol-regulated genes by probe set ranked highest to 224 Yan et al. Downloaded from jpet.aspetjournals.org

Fig. 2. Application of fractional, irreversible, b2-adrenoceptor inactivation to estimate pharmacodynamic parameters that define LABA-induced CRE reporter activation. 6CRE BEAS-2B reporter cells were treated with DCITC (100 nM) or vehicle for 60 minutes, washed in DCITC-free medium, and exposed to formoterol (A), indacaterol (B), salmeterol (C), picumeterol (D), and b2A (E). At 6 hours cells were lysed and luciferase activity was determined, from which E/[A] curves were constructed. The resulting pairs of curves were analyzed by operational model-fitting, from which estimates of KA, t, n, Em, and p[A]50 (of the control curve) were derived (Table 2). The horizontal dashed lines in panels A to E represent baseline luciferase activity. In at ASPET Journals on September 30, 2021 (F), the KA values of each agonist were used to calculate the relationship between fractional b2-adrenoceptor occupancy and reporter activation. The diagonal dashed line is the line of identity, at which luciferase activity is a linear function of receptor occupancy. Data represent the mean 6 S.E.M. of N independent determinations. lowest at 1, 2, and 6 hours. Thirteen induced genes and two probe set that produced the greatest change in expression per repressed genes were selected from the microarray and gene (Fig. 8A). validated by real-time PCR using the same RNA (Fig. 7). Ontological Analysis of Indacaterol-Regulated Genes. These are underlined in each heat map and their expression To gain information on the functional roles of genes that were level changes are given in each cell in log2 format (Supple- significantly up- and downregulated by indacaterol at 1, 2, and mental Figs. 2 and 3). PCR-validated probe sets that had 6 hours, functional annotation clustering was performed using changed by .1.5- or ,0.67-fold are also shown as yellow DAVID. These functional clusters were generated from the circles on each volcano plot in Fig. 4. For most genes, changes genes listed in Supplemental Tables 3 and 4, and are provided in expression measured by PCR were of greater magnitude in Supplemental Tables 5 and 6. than those determined by the microarray (Fig. 7) and this Indacaterol-Induced Genes at 1 Hour. Functional an- was confirmed by the finding that the slope of the line of notation of the 134 indacaterol-induced genes at 1 hour generated regression (1.15) deviated significantly from unity (Fig. 8A). 11 clusters of enriched GO terms (Supplemental Table 5). Clusters Nevertheless, a significant rank order correlation was found 1, 3, 4, 8, and 9 relate predominantly to gene regulation. The broad between the two formats after 2 hours of treatment using the descriptor nucleus (GO:0005634) associated with 41% of the gene

TABLE 2

Pharmacodynamic parameters that define b2-adrenoceptor-mediated, CRE-dependent reporter activation in BEAS-2B cells

Agonist E/[A] curves were constructed for cells treated with or without DCITC (100 nM; 60 min) and analyzed simultaneously by operational model-fitting. p[A]50 and p[A9]50 refer to the concentration of agonist that produced half maximum response in the absence and presence of DCITC, respectively. Parameters were derivedfromthedatainFig.2.

Parameter Estimates Treatment N p[A]50 p[A9]50 pKA KA/[A]50 KA/[A9]50 Emax (fold) Em (fold) n t b2A 7 8.71 6 0.04 7.34 6 0.06 7.20 6 0.07 32.5 1.41 12.9 6 0.7a 13.0 6 0.7 1.62 6 0.09 34.7 6 7.1 Formoterol 12 10.36 6 0.04 8.74 6 0.12 8.78 6 0.08 45.0 0.62 34.2 6 2.3 34.4 6 2.4 1.97 6 0.21 43.9 6 6.6 Indacaterol 12 8.61 6 0.05 7.19 6 0.11 6.98 6 0.07 46.1 1.25 33.8 6 2.1 34.1 6 2.1 1.37 6 0.11 47.2 6 6.8 Salmeterol 12 9.06 6 0.05 8.13 6 0.04 8.57 6 0.09 3.5 0.43 28.9 6 1.3 30.8 6 1.7 3.08 6 0.50 4.3 6 0.5 Picumeterol 12 9.63 6 0.07 8.83 6 0.08 9.24 6 0.13 3.2 0.48 27.0 6 1.9 29.2 6 2.3 2.75 6 0.37 4.1 6 0.6

a Maximum reporter drive varied between different batches of transfected cells and accounts for the lower Emax value of b2A (cf., b2A data in Table 1). Indacaterol-Regulated Transcriptome in Human Airway Epithelia 225 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2021

Fig. 3. Kinetics of indacaterol-induced gene expression changes. RNA was prepared from BEAS-2B cells treated for 1, 2, 6, and 18 hours with indacaterol (10 nM) or vehicle and subjected to gene expression profiling by microarray. Probe sets meeting the expression criteria (.1.5-fold, .3- fold, ,0.67-fold, ,0.33-fold) were analyzed using freely available software to identify DEGs that were significantly (FDR P value ,0.1) induced or repressed relative to time-matched, vehicle-treated cells. These are based on Entrez Gene and DAVID IDs and the number is shown in parentheses at each time-point. The pie charts represent the proportion of DEGs at each time-point that were induced (red) or repressed (blue). One gene, USP2 (probe set 229337_at), was apparently up- and downregulated by indacaterol, depending on the time of exposure. Note, this work forms part of a larger investigation that examined the effects of indacaterol and a phosphodiesterase 4 inhibitor, GSK 256066, alone and in combination, on gene expressionin human airway epithelial cells (see GSE106710). Accordingly, statistical analysis of data sets from all treatment groups was performed concurrentlyat each time-point. list and the more restricted but enriched terms negative regula- and 10.4% of the list, respectively; many of these genes encode tion of transcription from RNA polymerase II promoter (GO: sequence-specific transcription factors (e.g., ATF3, CEBPD, 0000122), positive regulation of transcription from RNA polymer- FOS2L, KLF2, KLF4, NFIL3, NR4A family members, TSC22D1) ase II promoter (GO:0045944), and transcription from RNA as well as several coactivators (e.g., CITED2) and corepressors polymerase II promoter (GO:0006366) comprised 17.2%, 19.4%, (e.g., FEZF, PRDM1, BCL6, BHLHE40). 226 Yan et al. Downloaded from

Fig. 4. Preliminary ontological characterization of indacaterol-induced gene expression changes. The relative expression patterning of probe sets at 1, 2, 6, and 18 hours was visualized by generating volcano plots (A–D, respectively). Each probe set is represented by a circle colored gray (transcript changes from .0.67-fold to ,1.5-fold), red or

blue (corresponding to transcripts that are induced or jpet.aspetjournals.org repressed by .1.5-fold and ,0.67-fold, respectively). Yellow circles represent DEGs that were validated by real-time PCR (Fig. 7). The vertical dashed line in each panel indicates baseline gene expression. NCBI Gene, GO, and Gene Cards were used to manually assign each gene to one of six broad groups: 1) transcriptional regulators (red), 2) transporters, ion channels and membrane receptors (orange), 3) metabolic proteins (yellow), 4) general cell

signaling components, including translational regulators at ASPET Journals on September 30, 2021 (green), 5) other functions (blue), and 6) not assigned (purple). These are represented as pie charts shown to the right (induced) and left (repressed) of each volcano plot, respectively.

The remaining six clusters include biologic process or of these terms is populated with genes that encode signaling molecular function descriptors that relate to inflammation molecules with proinflammatory potential (e.g., AREG, including cellular response to interleukin-1 (GO:0071347), BDNF, C5AR1, CTGF, CCL20, CXCL2, CXCL3, EDN1, IL6). cellular response to tumour necrosis factor (GO:0071356), Other genes in these clusters (e.g., ID1, MYC, NFIL3, NR4A3, cell-cell signalling (GO:0007267), response to lipopolysaccha- SOX9) encode transcriptional regulators involved in wound ride (GO:0032496), positive regulation of neutrophil chemo- healing, repair, airway hyper-responsiveness, and, poten- taxis (GO:0090023), and positive regulation of ERK1 and tially, remodeling and were annotated with the GO terms ERK2 cascades (GO:0070374) (Supplemental Table 5). Each positive regulation of epithelial cell proliferation (GO:0050679) Indacaterol-Regulated Transcriptome in Human Airway Epithelia 227 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2021

Fig. 5. Indacaterol-induced genes in BEAS-2B cells. Probes sets meeting the expression criteria (.1.5-fold induction; FDR P value ,0.1) were analyzed using Entrez Gene and the gene identification tool in DAVID to produce 134, 321, 205, and 9 upregulated genes at 1, 2, 6, and 18 hours, respectively. Induced genes that were subjected to independent PCR validation (Fig. 7) are in boldface and underlined. GO, NCBI Gene, and Gene Cards were used to manually assign each induced gene to one of six broad descriptors: 1) Transcriptional regulators, 2) Transporters, ion channels and membrane receptors, 3) Metabolic proteins, 4) General signaling molecules including translational regulators, 5) Other functions, and 6) Not assigned. Note: The following genes were detected with promiscuous probe sets (unannotated genes are not listed in the table): SIK1, LOC102724428 (208078_s_at, 232470_at), CHRNA7, LOC101929970, SLC24A2 (236385_at), AKIP1, NUAK2 (220987_s_at), SLC2A14, SLC2A3 (216236_s_at, 222088_s_at), HRK, LOC283454 (229552_at), ITGB6, LOC100505984 (208083_s_at), FAM87A, FAM87B (1559141_s_at, 1559140_at), CSNK1E, LOC400927-CSNK1E (222015_at, 226858_at), CREB5, LOC401317 (242329_at), RUNX1, LOC100506403 (210805_x_at) and UGT1A8, UGT1A9 (221305_s_at). 228 Yan et al. Downloaded from jpet.aspetjournals.org

Fig. 6. Indacaterol-repressed genes in BEAS-2B cells. Probes sets meeting the expression criteria (,0.67-fold repression; FDR P value ,0.1) were analyzed using the gene identification tool in DAVID to produce 46, 47, 47, and 1 downregulated genes at 1, 2, 6, and 18 hours, respectively. Repressed genes that were subjected to independent PCR validation (Fig. 7) are in boldface and underlined. GO, NCBI Gene, and Gene Cards were used to manually assign each repressed gene to one of the six broad terms described in Fig. 5. Note: The following genes were detected with promiscuous probe

sets (pseudogenes and unannotated genes are not listed in the table): NBPF1, NBPF8, NBPF9, NBPF10, NBPF11, NBPF12, NBPF14, NBPF19, at ASPET Journals on September 30, 2021 NBPF26, LOC100996763, LOC102724250 (1569519_at), KCTD14, NDUFC2-KCTD14 (219545_at), NPIPB4, NPIPB5, SMG1, SMG1P1, SMG1P2, SMG1P3, SMG1P5, SMG1P7, LOC101060596, LOC105369244, LOC105369278 (244766_at) and TPSAB1, TPSB2 (207134_x_at).

and positive regulation of smooth muscle cell proliferation (GO: IL11, IL15), chemokines (e.g., CCL2, CCL20, CXCL2), growth 0048661) (Supplemental Table 5). factors (e.g., AREG, CTGF, EDN1, FGF9, HBEGF), the Indacaterol-Induced Genes at 2 Hours. Twenty clus- neurotrophin BDNF, and the ephrin receptor tyrosine kinase ters of enriched terms were produced from the 321 genes that EFNA1. Enriched GO terms are also associated with genes were significantly induced by indacaterol at 2 hours (Sup- encoding proteins with therapeutic potential, such as CD200, plemental Table 5). The most highly enriched clusters (1–3) CRISPLD2, and DMBT1, which have antibacterial, antiviral, and cluster 11 contain terms that relate to DNA binding and/or anti-inflammatory activity. Clusters 4, 8, 9, 12, 14, and and transcription. In particular, 53 (16.5%) and 43 (13.4%) 15 contain many of the same genes present in cluster 6 and are upregulated genes were annotated with the GO terms enriched and annotated with a variety of more specific positive regulation of transcription from RNA polymerase II descriptors that are consistent with a proinflammatory phe- promoter (GO:0045944) and transcription factor activity, notype that includes growth factor activity (GO:0008083), sequence-specific DNA binding (GO:0003700), respectively. neutrophil chemotaxis (GO:0030593), cytokine activity (GO: Many of these genes encode sequence-specific transcrip- 0005125), TNF signalling pathway” (HSA:04668), monocyte tional activators (e.g., CEBPA, CEBPD, CREB3L2, FOS, chemotaxis (GO:0002548), and cellular response to transform- FOSB, FOSL2, FOXC1, FOXC2, FOXO1, HOXA5, KLF4, ing growth factor beta stimulus (GO:0071560) (Supplemental NR4A family members, NR2F2, TFAP2A, TRAP2C), coac- Table 5). tivators or corepressors (e.g., CITED4, VGLL2, ZBTB38). The remaining clusters contain genes with diverse activities Cluster 20 contains the term Iroquois-class homeodomain that are annotated with terms that relate to calcium, sodium, protein (IPR:003893), which is populated with genes encoding glucose, amino acid, monocarboxylic acid, thiamine, and transcription factors (i.e., IRX1, IRX2, IRX4) involved in carnitine transport (Clusters 7, 10 and 19), proteins covalently development. linked to the plasma membrane (Cluster 13), protein phos- The general GO terms integral component of plasma phorylation (Cluster 16), endoderm formation, protein de- membrane (GO:0005887), extracellular space (GO:0005615), phosphorylation and mitogen-activated protein (MAP) kinase and extracellular region (GO:0005576) are present in cluster inactivation (Cluster 18) and response to dexamethasone and 6 and associated with 45, 41, and 39 genes, respectively. These antibiotics (Cluster 17; Supplemental Table 5). genes encode solute carriers as well as many secreted proteins Indacaterol-Induced Genes at 6 Hours. Analysis of the with proinflammatory properties, such as cytokines (e.g., IL6, 205 indacaterol-induced genes at 6 hours generated eight Indacaterol-Regulated Transcriptome in Human Airway Epithelia 229 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2021

Fig. 7. Validation of indacaterol-induced gene expression changes. RNA was extracted from BEAS-2B cells treated for 1, 2, 6, and 18 hours with indacaterol (10 nM) or vehicle and subjected to gene expression profiling by microarray. Panels show the kinetics of 13 induced and two repressed genes and are plotted as fold change on a log2 scale, where a value of 1 indicates baseline gene expression (dashed horizontal lines). The open circles represent data taken directly from the microarray using the probe set that gave the greatest induction or repression relative to vehicle-treated, time-matched controls. These data were validated by PCR using the same RNA (see Supplemental Table 2 for primers) and are presented as a ratio to GAPDH at each time-point (filled black circles). Probe set IDs are shown within each panel. PCR data are the mean 6 S.E.M. of four determinations. 230 Yan et al.

Fig. 8. Relationships between LABA-induced gene expression changes in BEAS-2B cells. (A) The correlation at 2 hours between changes in the expression of 15 indacaterol-regulated genes in BEAS-2B cells measured by microarray (using the probe set that produced the greatest change relativeto vehicle) and PCR. The same RNA was used in both analyses. (B) The correlation of 12 gene expression changes determined by PCR in BEAS-2B cells Downloaded from stimulated for 2 hours with indacaterol (10 nM) and salmeterol (100 nM). Induced and repressed genes are shown as red and blue circles, respectively. Solid and dashed diagonal lines represent linear regression and the line of identity, respectively. In (A), Human Genome Nomenclature Committee gene symbols are shown together with Affymetrix probe set ID numbers from which the microarray data were derived. clusters of enriched terms (Supplemental Table 5). Clusters cell motility, growth, and differentiation. ADAMTSL1, GPC3 1 and 2 contain generic, cellular component descriptors such and SMOC1 were also part of this gene cluster but have as extracellular space (GO:0005615), integral component of unclear roles in airway epithelia. Genes associated with jpet.aspetjournals.org plasma membrane (GO:0005887), and plasma membrane (GO: cluster 8 were annotated by the KEGG term pathways in 0005887), and are populated with 13.2%, 15.6%, and 30.2% of cancer (HSA:05200) with representation from cell surface the gene list, respectively. Many genes associated with these receptors (BDKRB2, FGFR2, IGF1R, LPAR6), transcription terms are consistent with both beneficial and deleterious factors (CEBPA, FOXO1), endogenous FZR agonists (WNT4, effects of b2-adrenoceptor agonists in asthma. These include WNT5A), an E3 ubiquitin-protein ligase (MDM2), a type IV growth factor, tyrosine kinase-linked and G protein-coupled collagen (COL4A3), a junctional plaque protein (JUP), and an receptors (e.g., ACKR1, ADGRD2, AVPR2, BRKRB2, C5AR1, enzyme involved in apoptosis (CASP9). at ASPET Journals on September 30, 2021 CNR1, EPHA5, FGFR2, GAS1, GPRC5A, IGF1R, LPAR6), Indacaterol-Repressed Genes at 1 Hour. Functional cytokines, chemokines and related proteins (e.g., CCL2, IL20, annotation of the 46 indacaterol-repressed genes at 1 hour, IL1RAPL1), as well as a variety of secreted factors (e.g., generated a single cluster of descriptors (Supplemental CRISPLD2, CD200, DMBT1, WNT4, WNT5A). Several of Table 6). The most highly enriched GO term was DNA repair these genes (e.g., FGFR2, GAS1, WNT4, WNT5A) are also (GO:0006281) and comprised three genes (6.5% of the list): annotated by the more specific biochemical process GO CHEK1, a protein kinase necessary for checkpoint-mediated terms cell fate commitment (GO:0045165) and epithelial to cell cycle arrest and activation of DNA repair; RECQL5,a mesenchymal transition (GO:0001837), which are enriched in DNA helicase, which inhibits elongation of stalled transcripts cluster 3. at DNA damage sites; and EID3, a component of a complex Cluster 4 includes genes encoding cyclic nucleotide phos- involved in the repair of double-strand breaks in DNA. phodiesterases (PDE3A, PDE4D, PDE7B, PDE10A). These Indacaterol-Repressed Genes at 2 Hours. Two clusters are principally involved in cAMP degradation and were of enriched GO terms were generated from the 47 genes that annotated with the similar GO terms cAMP catabolic process were significantly repressed by indacaterol at 2 hours (Sup- (GO:0006198) and 39,59-cyclic-AMP phosphodiesterase activity plemental Table 6). Cluster 1 contains descriptors that relate (GO:0004115) (Supplemental Table 5). to mitotic cell division, in particular the GO biochemical Two of the remaining four clusters (5 and 7) contain genes process terms cell division (GO:0051301) and mitotic nuclear (SLC16A6, SLC16A14, SLC22A3, SLC22A4, SLC22A5) that division (GO:0007067). These were populated with genes that regulate transmembrane monocarboxylic acid and organic account for 9%–13% of the list, respectively, and include cation transport, and were annotated with the GO terms BUB1, BORA, CCNF, HMGA2, PARD6B, and PSRC1. quaternary ammonium group transport (GO:0015697) and Cluster 2 contains nine GO terms that are associated symporter activity (GO:0015293). In contrast, the most with DNA binding and the regulation of gene transcription. enriched GO term in cluster 6 was proteinaceous extracellular The most enriched term in this cluster, transcription, DNA matrix (GO:0005578). Genes within this group encode pro- templated (GO:0006351), was populated with 11 genes (23.4% teins with diverse functional roles, including: an LPS-binding of the list) that encode both established and putative tran- protein that can neutralize the pathogenicity of Gram nega- scription factors (EGR3, KLF7, SIM1, ZNF99, ZNF547, tive bacteria (CRISPLD2); two endogenous agonists (WNT4, ZNF552, ZNF556, ZNF594, ZNF738), a transcriptional coac- WNT5A) of the frizzled receptor (FZR) family of GPCRs; tivator (MED3L), and an inhibitor of thioredoxin 1 (TXNIP). surfactant protein D (SFTPD); a serine proteinase inhibitor Indacaterol-Repressed Genes at 6 Hours. Functional (TFPI2) implicated in the regulation of plasmin-mediated annotation of the 47 genes repressed at 6 hours generated a matrix remodeling; the a3-chain of type IV collagen (COL4A3); cluster of 11 enriched terms that relate to a host’s response to and a hyaluronan-binding protein (VCAN), which may control virus infection (Supplemental Table 6). Five genes (IFI44, Indacaterol-Regulated Transcriptome in Human Airway Epithelia 231 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 30, 2021

Fig. 9. Effect of salmeterol on gene expression in BEAS-2B cells and the effect of PKIa. Cells at ∼70% confluency were infected with Ad5.CMV.PKIa (PKI), Ad5.CMV.GFP (GFP, MOI = 25), or left untreated. After 48 hours, salmeterol (Salm; 100 nM) or vehicle was added for 2 hours and changes in the expression of 15 genes (ordered alphabetically) relative to GAPDH were determined by real-time PCR. Bars represent the mean 6 S.E.M. of N independent determinations. The dashed horizontal line in each panel represents baseline gene expression. *P , 0.05; **P , 0.05 indicate significant inhibition by PKIa of gene expression in cells treated with salmeterol and vehicle, respectively and was determined by repeated measures, ANOVA followed by Tukey multiple comparison test.

MX1, OAS1, OAS2, OAS3) were associated with the biochemical Despite its low operational efficacy, salmeterol increased process and molecular function GO descriptors type I interferon the expression of all genes studied at 2 hours relative to signaling pathway (GO:0060337), response to virus (GO:0009615), time-matched, vehicle-treated cells (Fig. 9). Moreover, there defence response to virus (GO:0051607), and 29,59-oligoadenylate was a significant, rank order correlation between the salmeterol- synthetase activity (GO:0001730), and encode proteins that block induced genes that were common with the PCR-validated the replication of RNA and some DNA viruses. indacaterol results at 2 hours shown in Fig. 7. Linear regression Effect of Salmeterol on Gene Expression in BEAS- yielded a slope of 0.84, which was not significantly different 2B Cells and the Effect of PKIa. To determine if the from unity, indicating that the magnitudes of many gene genomic effects of indacaterol extended to other LABAs, expression changes induced by both LABAs were comparable 15 induced genes were selected from the microarray and their (Fig. 8B). expression level changes (as a ratio to GAPDH) were de- The role of canonical cAMP signaling in mediating termined in BEAS-2B cells exposed for 2 hours to a maximally salmeterol-induced gene expression is also shown in Fig. 9. effective concentration of salmeterol (100 nM). Salmeterol was BEAS-2B cells were infected with Ad5.CMV.PKIa (MOI 5 25), selected for these experiments because it is a clinically which encodes a highly selective inhibitor of PKA (Takio et al., relevant, low efficacy LABA (cf., indacaterol; Table 2) and 1984; Glass et al., 1986), or a control vector, Ad5.CMV.GFP. Of can behave as a partial agonist and antagonist at b2-adreno- the 15 genes analyzed, five are proinflammatory (C5AR1, ceptors depending on the tissue or functional response of CXCL2, EDN1, IL6 and IL11), eight have putative disease- interest (Dougall et al., 1991; Rabe et al., 1993; Duringer et al., modifying activity (CD200, CDKN1C, CRISPLD2, DMBT1, 2009). DUSP1, GAS1, RGS2, and SOCS3), and the remaining two 232 Yan et al. Downloaded from jpet.aspetjournals.org

Fig. 10. Effect of salmeterol on gene expression in HBECs. Cells from five donors were exposed to vehicle (red circles) or salmeterol (black circles; at ASPET Journals on September 30, 2021 100 nM) for 2 and 6 hours (V2h, V6h, S2h and S6h, respectively). cDNA was prepared and subjected to real-time PCR using primers specific for 27 genes selected from the microarray (Supplemental Table 2). Changes in gene expression in cells harvested from each donor are presented relative to GAPDH and are ordered alphabetically. The mean salmeterol-induced fold change is shown at the top of each panel with the range in brackets. *P , 0.05, significant change from vehicle-treated cells, Student paired t test. NS, Not significant.

(NR4A2 and NR4A3) have unclear functions in the airways. significantly rank order correlated (Fig. 11A) and the line of Regardless of their (patho)physiologic roles, this intervention regression was not statistically different from a value of 1. prevented the induction of all 15 genes, indicating that This relationship was preserved if the expression levels of the signaling downstream of Gsa was the sole regulator of 27 genes in salmeterol-treated HBECs were correlated transcription (Fig. 9). against the corresponding changes in indacaterol-exposed PKIa also significantly reduced the basal expression BEAS-2B cells (Fig. 11B). However, in this comparison, of several of the 15 genes examined (Fig. 9). This was replicated discrepancies were apparent. In particular, the expression of in cells exposed to salmeterol (100 nM), which suggests that C5AR1, CDKN1C, CRISPLD2, DMBT1, IL11, NR4A1, and PKA was activated under resting (i.e., serum-free) conditions. NR4A3 was not statistically different from vehicle in HBECs The control vector, encoding GFP, was inactive. exposed to salmeterol, whereas in BEAS-2B cells they were Effect of Salmeterol on Gene Expression in HBECs. induced (Fig. 11). Other anomalies included the regulation of The effect of salmeterol (100 nM) on gene expression was CITED4 at 2 hours, which was increased and decreased by determined in HBECs harvested from the upper right or salmeterol in BEAS-2B and HBECs, respectively, and TXNIP upper left lung lobe of five male subjects who were 18–62 years whose expression was transiently reduced in BEAS-2B cells of age at the time of death (see Supplemental Table 1 for yet increased in HBECs at 6 hours. demographics). These genes were randomly selected from the BEAS-2B microarray data as being either significantly in- Discussion duced or repressed by indacaterol at either 2 or 6 hours (FDR P value , 0.1). Inhaled b2-adrenoceptor agonists were introduced for the Figure 10 shows gene expression (as a ratio to GAPDH) for treatment of asthma in 1968, and since that time ASM each donor at baseline and after 2- and 6-hour exposures to relaxation has been assumed to account for their clinical salmeterol. Consistent with indacaterol microarray data, the efficacy (Sneader, 2001). The increase in asthma mortality majority of these genes were correspondingly induced or reported in the 1960s and 1970s with isoprenaline and repressed by salmeterol in HBECs at one or both time- fenoterol given chronically as a monotherapy (Cockcroft and points. Indeed, by PCR at 2 hours, changes in the expression Sears, 2013) can also be attributed to long-lasting bronchodi- of a panel of genes in HBECs and BEAS-2B cells were latation, because the underlying inflammation would have Indacaterol-Regulated Transcriptome in Human Airway Epithelia 233

Fig. 11. Concordance of gene expression changes in BEAS-2B cells and HBECs. (A) Relationship between the effects of salmeterol (100 nM for 2 hours) on the expression in HBECs of 20 of the genes shown in Fig. 10 with their counterparts in BEAS-2B cells measured by PCR. (B) A similar plot between all gene expression changes measured in HBECs exposed to salmeterol (Fig. 10) and their indacaterol (10 nM)-stimulated equivalents in BEAS-2B cells (using probe sets that produced the greatest change relative to vehicle). Induced and repressed genes are shown in red and blue, respectively. Solid and dashed diagonal lines represent linear regression and the line of identity, respectively. In (B), Human Genome Nomenclature Committee gene symbols Downloaded from are indicated together with Affymetrix probe set ID numbers from which the microarray data were derived.

been masked and resulted, theoretically, in a loss of disease More than 40 genes involved in transcriptional control were control (McIvor et al., 1998). However, the discovery that induced by indacaterol in BEAS-2B cells and annotated with indacaterol changed the expression of hundreds of genes in the highly enriched GO term positive regulation of transcription jpet.aspetjournals.org BEAS-2B cells suggests that the adverse and beneficial effects from RNA polymerase II promoter. Although several of these of b2-adrenoceptor agonists in asthma may extend beyond genes encode repressors, the majority are positive regulators of ASM relaxation and involve genomic mechanisms that oper- transcription. Thus, several FOS transcripts (FOS, FOSB and ate in a variety of airway cells including the epithelium. FOSL2), whose products dimerize with c-Jun family members Functional annotation clustering of the indacaterol-regulated to form the transcription factor AP1, were significantly upregu- transcriptome implicates populations of genes that can exac- lated. The expression of NFIL3 and CEBPD were similarly erbate airway pathology and may contribute to the reported increased. NFIL3 promotes IgE class switching, airway hyper- at ASPET Journals on September 30, 2021 toxicity of regular b2-adrenoceptor agonist monotherapy. responsiveness (Kashiwada et al., 2010), macrophage activa- Likewise, other genes were identified that may exert thera- tion, CD41 T-cell polarization and the development of natural peutic benefit, especially when used in combination with an killer and dendritic cells (Male et al., 2012). In contrast, CEBPD ICS (vide infra), by exerting antiviral, antibacterial, anti- regulates immune and inflammatory responses by promoting inflammatory, and, possibly, anti-airway remodeling activi- the transcription of proinflammatory cytokine genes, including ties. The replication of many key gene expression changes in IL6 (Kinoshita et al., 1992), whose expression was also in- HBECs in response to the low efficacy agonist salmeterol creased in indacaterol-treated BEAS-2B cells. indicates that this genomic effect is not peculiar to the BEAS- Indacaterol-Induced Genes with Therapeutic Poten- 2B cell line and may be shared by other clinically used SABAs tial. The indacaterol-induced transcriptome likewise con- and LABAs. tains genes with therapeutic potential. Generally, their Indacaterol-Induced, Adverse-Effect Genes. A strik- functions are poorly described and annotated with broad ing feature of the indacaterol-induced transcriptome was that cellular component GO terms such as nucleus, extracellular many enriched GO terms, including neutrophil chemotaxis, space, and secreted. Nevertheless, manual curation identified cellular response to interleukin 1, and response to lipopolysac- many genes (e.g., DUSP1, SOCS3, CD200) with predicted charide were populated with genes that can generate a therapeutic activity on the basis of in vitro observations and proinflammatory phenotype. Cytokine, chemokine, and studies in preclinical models of inflammation. DUSP1 is a growth factor mRNAs were significantly upregulated by phosphatase that catalyzes the inactivation of the three core indacaterol. These included CCL2, CCL20, CXCL2, and MAP kinases: extracellular signal-regulated kinase, p38 MAP CXCL3, which encode proteins that are chemotactic for kinase, and c-Jun-N-terminal kinase (Liu et al., 2007), which neutrophils, hematopoietic stem cells, monocytes, memory are central regulators of virtually all aspects of inflammatory T-cells, dendritic cells and lymphocytes (Wolpe et al., 1989; processes, including gene expression (Kyriakis and Avruch, Carr et al., 1994; Xu et al., 1996; Hieshima et al., 1997; Smith 2012). SOCS3 negatively regulates IL-6-, IL-12-, and G-CSF- et al., 2005; Pelus and Fukuda, 2006). Similarly, BDNF, a signaling by inhibiting the JAK/STAT pathway (Yoshimura neurotrophin that maintains the survival and activation of et al., 2007), whereas CD200 interacts with CD200R-bearing immune and structural cells in the lung and is elevated in the cells, such as macrophages, to suppress proinflammatory cyto- sputum of subjects with severe asthma (Watanabe et al., kine generation (Snelgrove et al., 2008); it may also attenuate 2015), was also induced by indacaterol. This finding is airway hyperresponsiveness (Lauzon-Joset et al., 2015). consistent with the ability of salmeterol to upregulate BDNF Other genes were identified that could protect against bacteria- in the blood of patients with mild asthma (Lommatzsch et al., and virus-induced exacerbations of asthma. CRISPLD2,which 2009), implying that SABA and LABA monotherapy may was originally implicated in the etiology of nonsyndromic cleft enhance pre-existing inflammation. lip (Chiquet et al., 2007), encodes a secreted, LPS-binding 234 Yan et al. protein in both humans and mice (Wang et al., 2009). Evidence for the inconsistencies, gene expression changes were signif- that it can neutralize the pathogenicity of Gram negative icantly rank-order correlated and preserved when the salme- bacteria by downregulating TLR4-mediated proinflammatory terol PCR results in BEAS-2B cells were substituted with the responses is available (Zhang et al., 2016). Similarly, DMBT1 corresponding indacaterol data from the array. Thus, a similar encodes a multifunctional, secreted protein that is present in genomic impact of indacaterol and salmeterol on HBECs and human bronchoalveolar lavage fluid (Holmskov et al., 1997). BEAS-2B cells is implied. Through its ability to interact with a range of polysulfated and b2-Adrenoceptor-Mediated Toxicity and the Effect of polyphosphorylated ligands, DMBT1 binds Gram positive and ICSs. The results presented herein could explain why regular Gram negative bacteria and may, therefore, provide a defense SABA and LABA monotherapy makes asthma worse. Scrutiny against microbial pathogens (Madsen et al., 2010). DMBT1 of the gene expression fingerprint of indacaterol in BEAS- also inhibits the infectivity of human influenza A and 2B cells identified hundreds of DEGs. While some of these immunodeficiency viruses (Madsen et al., 2010). encode proteins with putative therapeutic benefit, there were Of the indacaterol-induced genes involved in transcriptional arguably many more that could promote, or even exacerbate, control, NR4A1, NR4A2, and NR4A3 were markedly upregu- airways inflammation (vide supra). Thus, the net effect of lated. There is emerging evidence that these early response regular SABA and LABA monotherapy could increase asthma genes, which encode ligand-independent transcription factors, severity and enhance mortality by preferentially enhancing function as negative feedback regulators of inflammation proinflammatory gene expression. A genomic effect is also (Rodriguez-Calvo et al., 2017). In the immune system, they germane to the mechanism of action of ICS/LABA combination Downloaded from are implicated in the maturation and/or development of therapy, because glucocorticoids repress the expression of monocytes, regulatory T cells, and CD81 lymphocytes. More- many of these adverse-effect genes (Newton and Giembycz, over, in Nr4a2-deficient mice, macrophages are polarized 2016) and protect against LABA-induced toxicity in asthmatic toward a proinflammatory M1 phenotype (Rodriguez-Calvo subjects (Cates et al., 2014). As a result, an ICS may tip the et al., 2017). Currently, the genes under the control of NR4A balance of a LABA away from side effects potentially toward family members are ill-defined. therapeutic benefit. jpet.aspetjournals.org Indacaterol-Repressed Genes. Depending on the time of An ICS can also augment, in an additive or synergistic exposure, 13%–26% of the indacaterol-regulated transcriptome manner, the expression of many LABA-induced genes that was repressed by ,0.67-fold. Notable genes that, when down- could be therapeutically desirable (Giembycz and Newton, regulated, may be beneficial include the transcription factor 2015; Newton and Giembycz, 2016). A recent microarray- EGR1, which is implicated in the etiology of airway fibrosis, based study using human airway epithelial cells identified inflammation, and remodeling (Lee et al., 2004; Cho et al., 2006). many genes that are regulated in this way, including

The ability of indacaterol to reduce the expression of TXNIP may CRISPLD2, RGS2, and DUSP1 (Rider et al., under review). at ASPET Journals on September 30, 2021 also be favorable. This gene encodes a protein that inhibits the Collectively, therefore, the ability of an ICS to repress and ability of thioredoxin 1 to reduce oxidized proteins. Accordingly, enhance, respectively, populations of LABA-inducible genes a reduction in TXNIP should enhance thioredoxin 1 activity and that mediate deleterious and beneficial effects on airway may reduce oxidative stress (Nishiyama et al., 1999). function may help explain why ICS/LABA combination ther- Repressed genes that may lead to undesirable outcomes apy often controls subjects with moderate-to-severe asthma, were also identified. These include OAS1, OAS2, and whereas ICS monotherapy does not (Giembycz and Newton, OAS3, which encode 29,59-oligoadenylates that degrade 2015; Newton and Giembycz, 2016). viral RNA and inhibit viral replication by activating latent Role of Canonical cAMP Signaling. Previous studies RNase L (Kristiansen et al., 2011). Two other antiviral have established that SABA- and LABA-induced gene expression genes (IFI44, MX1) were also repressed, suggesting that changes in BEAS-2B cells are mediated by the b2-adrenoceptor b2-adrenoceptor agonist monotherapy could compromise (Holden et al., 2014; Joshi et al., 2015, 2017; Kaur et al., 2008). the body’s innate response to infection (cf., CRISPLD2 and The ability of PKIa to prevent the induction of several DMBT1,videsupra). salmeterol-induced genes that are associated with enriched GO Gene Regulation in BEAS-2B Cells and HBECs by terms related to proinflammatory or therapeutic processes ex- Low Efficacy LABAs. Agonist intrinsic efficacy can vary tends those findings and provides evidence that cAMP/PKA markedly with profound functional consequences in cells signaling is a major regulator of gene transcription. Knowledge expressing a low receptor number or when receptor-effector that these gene promoters, and many more, feature cis-acting coupling is weak. The ability of salmeterol to behave as a full CREs for the transcription factor cAMP response element–binding agonist on ASM and an antagonist on eosinophils are extreme protein (CREB) (Zhang et al., 2005)—an established substrate examples of such behavior (Rabe et al., 1993; Munoz et al., for PKA—is consistent with this suggestion. However, inter- 1995; Aparici et al., 2012). In this study, salmeterol activated pretation of these data must be weighed against the fact that the CRE reporter and promoted gene expression changes that PKIa was examined on only 15 salmeterol-induced genes. Thus, were indistinguishable from those of indacaterol, despite the the role of alternative (e.g., arrestin-dependent) mechanisms of 10-fold difference in operational efficacy, indicating that b2- gene regulation cannot be excluded. Indeed, there is evidence adrenoceptor density on BEAS-2B cells was not limiting. that the b2-adrenoceptor can recruit Gs-independent pathways Salmeterol also promoted gene expression changes in through biased agonism (Rajagopal et al., 2011; van der West- HBECs. Although many of these were similarly regulated in huizen et al., 2014), suggesting that we may have unintention- BEAS-2B cells, discrepancies were apparent. These may be ally selected genes from the microarray that are transcribed, in explained by weak statistical power (see DMBT1 and NR4A3 response to salmeterol, in a PKA-dependent manner. in Fig. 10), intrinsic differences in gene regulation between the Limitations of the Work. A shortcoming that should be two cell types, or dissimilar culture conditions. Even allowing addressed in future studies is to establish the extent to which Indacaterol-Regulated Transcriptome in Human Airway Epithelia 235

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Supplemental Material

Analysis of the Indacaterol-Regulated Transcriptome in Human Airway Epithelial Cells Implicates Gene Expression Changes in the Adverse and Therapeutic Effects of β2-Adrenoceptor Agonists

Dong Yan, Omar Hamed, Taruna Joshi, Mahmoud M. Mostafa, Kyla C. Jamieson, Radhika Joshi, Robert Newton & Mark A. Giembycz

Departments of Physiology & Pharmacology (D.Y., O.H., K.C.J., T.J., R.J., M.A.G.) and Cell Biology & Anatomy (R.N., M.M.M.), Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.

(A)

Induced 120 Transcription 100 Receptors

t Signalling ) s n 80 Metabolic u D I o Other Functions C D I 60

e Not Assigned V n A e D ( G 40 20 0 0 1 2 6 18 Time (h) (B) Repressed 0

5 t ) s n u D I o C D I 10 e V n

A Transcription e D ( G Receptors 15 Signalling Metabolic Other Functions 20 Not Assigned 0 1 2 6 18 Time (h)

Supplemental Fig. 1. Effect of indacaterol on the number of significantly induced and repressed genes (panels A and B respectively) at 1h, 2h, 6h and 18h that were annotated with one of the six broad GO terms used in figure 4. Indacaterol (10nM) Indacaterol (10nM) Indacaterol (10nM) Top 100 Transcripts >1.5-fold Induced at 1h Top 100 Transcripts >1.5-fold Induced at 2h Top 100 Transcripts >1.5-fold Induced at 6h Affy ID 1h 2h 6h 18h Gene Affy ID 1h 2h 6h 18h Gene Affy ID 1h 2h 6h 18h Gene 204621_s_at 4.7 4.6 1.5 0.3 NR4A2 209959_at NR4A3 209959_at NR4A3 7 207978_s_at NR4A3 207978_s_at NR4A3 221541_at 0.3 3.1 3.0 0.7 CRISPLD2 216248_s_at 4.4 4.8 1.8 0.2 NR4A2 216248_s_at 4.4 4.8 1.8 0.1 NR4A2 1565657_at CMTM6 204622_x_at 4.4 4.6 1.5 0.3 NR4A2 204622_x_at 4.4 4.6 1.5 0.4 NR4A2 219049_at CSGALNACT1 209959_at NR4A3 204621_s_at 4.7 4.6 1.5 0.3 NR4A2 228128_x_at PAPPA 205207_at IL6 203639_s_at 0.4 3.8 1.6 -0.5 FGFR2 222784_at SMOC1 205476_at CCL20 205289_at BMP2 225207_at 1.92 3.05 2.44 0.77 PDK4 239145_at ZNF414 221541_at 0.3 3.1 3.0 0.7 CRISPLD2 239461_at GALNT15 228964_at PRDM1 225207_at 1.9 3.1 2.4 0.8 PDK4 222783_s_at SMOC1 208078_s_at SIK1 205476_at CCL20 243438_at PDE7B 209774_x_at CXCL2 205207_at IL6 205421_at SLC22A3 233972_s_at FEZF2 234511_at ANKRD60 1556185_a_at STEAP4 239818_x_at TRIB1 205960_at 2.1 2.7 1.8 0.5 PDK4 224942_at PAPPA 232470_at SIK1 232470_at SIK1 224940_s_at PAPPA 238378_at GPRIN3 208078_s_at SIK1 203638_s_at 0.8 2.5 2.2 0.5 FGFR2 202241_at TRIB1 218541_s_at C8orf4 224941_at PAPPA 201739_at 2.1 2.5 0.6 0.0 SGK1 228962_at 1.1 2.6 2.2 0.4 PDE4D 228962_at 1.0 2.6 2.2 0.4 PDE4D 205960_at 2.1 2.8 1.8 0.5 PDK4 201739_at 2.1 2.5 0.6 0.0 SGK1 230109_at PDE7B 202388_at 2.1 2.2 0.9 0.2 RGS2 206359_at 0.8 2.5 1.0 0.1 SOCS3 201981_at PAPPA 1557285_at AREG 209582_s_at 1.1 2.5 1.1 0.1 CD200 238649_at PITPNC1 225207_at 1.9 3.1 2.4 0.8 PDK4 203638_s_at 0.8 2.5 2.2 0.5 FGFR2 228501_at GALNT15 218995_s_at EDN1 228964_at PRDM1 203549_s_at LPL 201044_x_at 1.8 1.3 0.5 0.0 DUSP1 211401_s_at -0.1 2.4 2.0 1.3 FGFR2 211401_s_at -0.1 2.4 1.9 1.3 FGFR2 222802_at EDN1 204491_at 1.1 2.4 1.5 0.4 PDE4D 203548_s_at LPL 1555585_a_at FAM71B 227697_at 0.9 2.3 0.9 0.1 SOCS3 213050_at COBL 227613_at ZNF331 225582_at ITPRIP 220088_at 1.2 2.1 1.9 0.1 C5AR1 201169_s_at BHLHE40 210837_s_at 0.7 2.3 1.0 0.3 PDE4D 225987_at STEAP4 202340_x_at NR4A1 202388_at 1.4 2.3 0.7 0.1 RGS2 211119_at ESR2 211143_x_at NR4A1 210836_x_at 0.8 2.2 1.3 0.3 PDE4D 236361_at GALNT15 213139_at SNAI2 244025_at ITPRIP 237804_at DNAH11 219228_at ZNF331 205290_s_at BMP2 205960_at 2.1 2.8 1.8 0.5 PDK4 244025_at ITPRIP 209774_x_at CXCL2 229709_at ATP1B3 201170_s_at BHLHE40 205330_at MN1 216248_s_at 0.0 1.9 1.8 0.2 NR4A2 224797_at ARRDC3 1562321_at 1.8 2.1 1.1 1.1 PDK4 225516_at SLC7A2 205764_at CSNK1A1 230748_at SLC16A6 208606_s_at WNT4 203395_s_at HES1 209583_s_at 1.1 2.1 1.2 0.4 CD200 213182_x_at CDKN1C 221841_s_at KLF4 227613_at ZNF331 205501_at PDE10A 225582_at ITPRIP 207038_at SLC16A6 220540_at KCNK15 203394_s_at HES1 220088_at 1.2 2.1 1.9 0.1 C5AR1 219093_at PID1 239530_at ADD2 211143_x_at NR4A1 204491_at 1.1 2.4 1.5 0.3 PDE4D 238987_at B4GALT1 219228_at ZNF331 219534_x_at CDKN1C 205239_at AREG 229552_at HRK 216894_x_at CDKN1C 240413_at PYHIN1 239367_at BDNF 213348_at CDKN1C 203313_s_at TGIF1 208083_s_at ITGB6 220187_at STEAP4 210632_s_at SGCA 213182_x_at CDKN1C 223484_at C15orf48 227337_at ANKRD37 202340_x_at NR4A1 239650_at NCKAP5 220088_at 1.2 2.1 1.9 0.1 C5AR1 219911_s_at SLCO4A1 237939_at EPHA5 36711_at MAFF 1559141_s_at FAM87A, FAM87B 230748_at SLC16A6 205290_s_at BMP2 219534_x_at CDKN1C 207038_at SLC16A6 1559987_at MSI2 224828_at CPEB4 222015_at CSNK1E 201939_at PLK2 229709_at ATP1B3 216627_s_at B4GALT1 209582_s_at 1.1 0.8 0.0 -0.2 CD200 206382_s_at BDNF 210102_at VWA5A 1566901_at TGIF1 219682_s_at TBX3 229093_at NOS3 202935_s_at SOX9 203549_s_at LPL 228132_at ABLIM2 242543_at SH2D6 224829_at CPEB4 236300_at PDE3A 205193_at MAFF 216894_x_at CDKN1C 238029_s_at SLC16A14 229152_at FDCSP 206865_at HRK 210836_x_at 0.1 0.8 1.2 0.4 PDE4D 1555118_at ENTPD3 239818_x_at TRIB1 1555007_s_at WDR66 209583_s_at CD200 218177_at CHMP1B 228507_at PDE3A 204011_at SPRY2 218995_s_at EDN1 208228_s_at 0.3 1.5 1.2 0.0 FGFR2 204491_at 1.1 2.4 1.5 0.3 PDE4D 1552755_at C9orf66 209583_s_at 1.1 2.1 1.2 0.3 CD200 239367_at BDNF 216236_s_at SLC2A3, SLC2A14 229414_at PITPNC1 220125_at DNAI1 213348_at CDKN1C 1553199_at DCAF4L2 228962_at 1.0 2.6 2.2 0.4 PDE4D 213006_at CEBPD 224071_at IL20 204456_s_at 1.0 1.5 0.7 0.4 GAS1 202241_at TRIB1 225978_at RIMKLB 205466_s_at HS3ST1 209631_s_at GPR37 1555006_at WDR66 203574_at NFIL3 202497_x_at SLC2A3 221305_s_at UGT1A8, UGTA9 218177_at CHMP1B 228128_x_at PAPPA 229552_at HRK 209457_at DUSP5 201981_at PAPPA 208425_s_at TANC2 216604_s_at SLC7A8 203548_s_at LPL 209582_s_at -0.9 -0.7 1.2 0.1 CD200 212977_at ACKR3 224831_at CPEB4 232267_at ADGRD1 205027_s_at MAP3K8 222088_s_at SLC2A3, SLC2A14 206616_s_at ADAM22 206758_at EDN2 228325_at SPIDR 1552736_a_at NETO1 206382_s_at BDNF 220483_s_at RNF19A 1554199_at PTPRO 230748_at SLC16A6 202499_s_at SLC2A3 218177_at CHMP1B 207826_s_at ID3 224942_at PAPPA 205003_at DOCK4 206706_at NTF3 202498_s_at SLC2A3 227099_s_at C11orf96 218631_at AVPI1 224940_s_at PAPPA 206389_s_at PDE3A 218178_s_at CHMP1B 239123_at TSC22D1 209772_s_at CD24 202936_s_at SOX9 218178_s_at CHMP1B 206356_s_at GNAL 236385_at CHRNA7, CHRFAM7A 203984_s_at CASP9 218589_at LPAR6 229110_at SLC24A2 222802_at EDN1 228325_at SPIDR 202672_s_at ATF3 226614_s_at FAM167A 235367_at MYPN 208937_s_at ID1 205027_s_at MAP3K8 213006_at CEBPD 218810_at ZC3H12A 204456_s_at 1.1 1.5 0.8 0.4 GAS1 224952_at TANC2 208960_s_at KLF6 212226_s_at PLPP3 203628_at IGF1R 202023_at EFNA1 218723_s_at RGCC 213436_at CNR1 0 215111_s_at TSC22D1 212977_at ACKR3 218865_at MARC1 218723_s_at RGCC 208228_s_at 0.3 1.5 1.2 0.0 FGFR2 207626_s_at SLC7A2 239058_at FOXC2 225516_at SLC7A2 220335_x_at CES3 232746_at ACKR3 209355_s_at PLPP3 205157_s_at KRT17 225557_at CSRNP1 225544_at TBX3 230398_at TNS4 207850_at CXCL3 230472_at IRX1 205896_at SLC22A4 234583_at CHL1 210775_x_at CASP9 208078_s_at SIK1 1552948_at CCDC27 237187_at HRK 208651_x_at CD24 220987_s_at AKIP1, NUAK2 224941_at PAPPA 1568949_at PITPNC1 202149_at NEDD9 225606_at BCL2L11 206359_at 0.8 2.5 1.0 0.2 SOCS3 202431_s_at MYC 219316_s_at FLVCR2 207430_s_at MSMB 220266_s_at KLF4 228340_at TLE3 208650_s_at CD24 205815_at REG3A 1553613_s_at FOXC1 201810_s_at SH3BP5  -1 208250_s_at 0.3 1.3 0.9 0.4 DMBT1

Supplemental Fig. 2. Heats maps showing the effect of indacaterol on gene induction in BEAS-2B cells treated with indacaterol (10nM) for 1h, 2h and 6h. The top induced transcripts by probe set that were statistically different from time-matched, vehicle-treated cells at each time-point (FDR P -value <0.1) were ranked highest to lowest and presented as heat maps relative to changes of the same transcripts at the other two time-points. The colour scale is logarithmic (base 2) with the most intense red and blue colours representing a 128-fold increase (log2 = 7) and an 2-fold decrease (log2 = -1) in gene expression respectively. The right hand-side and left hand-side of every row in each heat map shows the gene name and corresponding Affymetrix identification number (Affy ID). Genes validated by PCR are underlined and bolded in black, and their expression level changes are shown in each cell in log2 format. At 2h, DMBT1 was the 128th highest transcript induced by indacaterol. Indacaterol (10nM) Indacaterol (10nM) Indacaterol (10nM) Top Transcripts <0.67-fold Repressed at 1h Top Transcripts <0.67-fold Repressed at 2h Top Transcripts <0.67-fold Repressed at 6h Affy ID 1h 2h 6h 18h Gene Affy ID 1h 2h 6h 18h Gene Affy ID 1h 2h 6h 18h Gene 210919_at PHLPP1 232328_at ZNF552 231600_at CLEC12B 3 1569262_x_at UBE3D 216345_at ZSWIM8 204823_at NAV3 236811_at DMRTC2 1569262_x_at UBE3D 207134_x_at TPSAB1 236862_at GOPC 227404_s_at -0.6 -1.4 -0.9 0.0 EGR1 1552658_a_at NAV3 237943_at TMCC1 201009_s_at -0.7 -1.3 0.1 0.1 TXNIP 217262_s_at CELSR1 237469_at TOP2A 230673_at PKHD1L1 223204_at FAM198B 218657_at RAPGEFL1 201694_s_at -0.6 -1.3 -1.0 -0.2 EGR1 223317_at ALKBH7 1557852_at PHC2 201008_s_at -0.6 -1.3 0.0 0.1 TXNIP 214978_s_at PPFIA4 1558124_at NUDCD2 1556300_s_at SIM1 1552546_a_at LETM2 1561720_at RECQL5 201010_s_at -0.6 -1.2 0.0 0.0 TXNIP 219872_at FAM198B 244427_at KIF23 206876_at SIM1 231053_at KCNB1 201693_s_at -0.8 -0.9 -0.5 -0.1 EGR1 237469_at TOP2A 201694_s_at -0.6 -1.3 -1.0 -0.2 EGR1 236841_at WASHC1 244427_at KIF23 218864_at TNS1 220084_at CCDC198 1555591_at PIF1 204439_at IFI44L 239194_at DNAJC30 216109_at MED13L 227404_s_at -0.6 -1.4 -0.9 0.0 EGR1 201009_s_at -0.7 -1.3 0.1 0.1 TXNIP 1566111_at PIEZO1 202086_at MX1 214918_at HNRNPM 206115_at EGR3 214453_s_at IFI44 240452_at GSPT1 1552880_at SEC16B 206757_at PDE5A 228455_at RBM15 215175_at PCNX 211006_s_at KCNB1 230998_at CBX3 215508_at BUB1 215649_s_at MVK 231292_at EID3 204826_at CCNF 204363_at F3 228393_s_at ZNF302 240432_x_at KLF7 223749_at C1QTNF2 242922_at NOMO3 1553883_at ZNF99 206553_at OAS2 1569519_at NBPF1 201693_s_at -0.8 -0.9 -0.5 -0.1 EGR1 229337_at USP2 1561965_at SNRPB2 238049_at GRAMD3 1556361_s_at ANKRD13C 1570173_at INTS7 232735_at ANKRD34A 220381_at ARHGAP28 242111_at METTL3 208025_s_at HMGA2 205552_s_at OAS1 201008_s_at -0.6 -1.3 0.0 0.1 TXNIP 232912_at GPR180 222071_s_at SLCO4C1 229423_at CHEK1 220709_at ZNF556 232666_at OAS3 1559993_at SFXN3 244766_at NPIPB4 233234_at KCTD16 1565703_at SMAD4 219544_at BORA 206825_at OXTR 201466_s_at JUN 228892_at SH3RF2 211520_s_at GRIA1  232055_at SFXN1 207286_at CEP135 233907_s_at SERTAD4 239044_at ITFG1 227687_at HYLS1 235315_at TSC22D1 201694_s_at -0.6 -1.3 -1.0 -0.2 EGR1 201896_s_at PSRC1 237767_at FARP1 239071_at RBBP4 230462_at NUMB 204938_s_at PLN 206683_at ZNF165 218192_at IP6K2 204056_s_at MVK 219545_at KCTD14 238220_at KDM6A 237691_x_at ENO3 1553349_at ARID2 1552546_a_at LETM2 1559060_a_at FNIP1 235690_at ZNF594 1554018_at GPNMB 216061_x_at PDGFB 1559449_a_at ZNF254 227801_at TRIM59 240869_at KCNB1 218726_at HJURP 226603_at SAMD9L 216081_at LAMA4 211018_at LSS 204349_at MED7 211019_s_at LSS 235165_at PARD6B 227055_at METTL7B 233782_at DCAF7 235544_x_at FAM171A2 235476_at TRIM59 236344_at PDE1C 214918_at HNRNPM 230708_at PRICKLE1 225283_at ARRDC4 235760_at NSD1 1552794_a_at ZNF547 229767_at TBCD 2

Supplemental Fig. 3. Heats maps showing the effect of indacaterol on gene repression in BEAS-2B cells treated with indacaterol (10nM) for 1h, 2h and 6h. The top repressed transcripts by probe set that were statistically different from time-matched, vehicle-treated cells at each time-point (FDR P value <0.1) were ranked highest to lowest and presented as heat maps relative to changes of the same transcripts at the other two time-points. The colour scale is logarithmic (base 2) with the most intense blue and red colours representing a 8-fold decrease (log2 = -3) and an 4-fold increase (log2 = 2) in gene expression respectively. The right hand-side and left hand-side of every row in each heat map shows the gene name and corresponding Affymetrix identification number (Affy ID). Genes validated by PCR are underlined and bolded in black, and their expression level changes are shown in each cell in log2 format.

Supplemental TABLE 1. Donor demographics.

Source of Donor ID# Age Gender Ethnicity Smoker Cause of Death Epithelial Cells p42 18 Male Caucasian No Head Trauma Right Lung p48 22 Male Caucasian Yes Anoxia Left Lung p72 62 Male Caucasian No Intracerebral Haemorrhage Right Lung p78 60 Male Hispanic Yes Head Trauma Left Lung p82 42 Male Caucasian Yes Intracerebral Haemorrhage Right Lung

Supplemental TABLE 2. Primer pairs for real-time PCR. Official HGNC gene symbols are shown. When appropriate common gene symbols are provided in parentheses. Generic primers were used for genes that encode multiple isoforms.

Gene Oligonucleotides Accession Number(s) AVPI1 NM_021732.2 Forward 5’-AAGGCTGTGGAAGAGGTTGG-3’ Reverse 5’-TGTGGATGCTCACACTGAAGG-3’ BDNF NM_001352269.1, NM_001352270.1, NM_001352267.1, NM_001352271, Forward 5’-GGCGGCAGACAAAAAGACTG-3’ NM_020734.4, NM_001352268.1, NM_001297776.1, XM_017019690.1, Reverse 5’-TGGGATTGCACTTGGTCTCG-3’ XM_017019683.1, XM_017019684.1, XM_017019685.1, XM_017019686.1 XM_017019687.1, XM_017019688.1, XM_017019689.1

C5AR1 NM_001736.3 Forward 5’-TCCTCCGGAACGTGTTGACT-3’ Reverse 5’-AGCGCGTGAATGACTTGCT-3’ CD200 NM_005944.5, NM_001004196.2 Forward 5’-GGACTGTGACCGACTTTAAGCAA-3’ Reverse 5’-AGCAATAGCGGAACTGAAAACC-3’ kip2 CDKN1C (p57P )P NM_000076.2, NM_001122630.1, NM_001122631.1 Forward 5’-CTGTCCGGGCCTCTGATCT-3’ Reverse 5’-CATCGCCCGACGACTTCT-3’ CEBPD NM_005195 Forward 5’-GGAGATGCAGCAGAAGTTGGT-3’ Reverse 5’-CGCGCTGGTGCAGCTT-3’ DEPDC7 NM_139160.2, NM_001077242.1 Forward 5’-GACTCTCAGGAAGATGAGTGG-3’ Reverse 5’-GTCTGGTTGCTCAGGAAAGC-3’ DMBT1 NM_004406.2, NM_007329.2, NM_017579.2, NM_001320644.1 Forward 5’-GGTGGCATCCAACAACTACAA-3’ Reverse 5’-GTAGGATGGGCTGGAGAATGT-3’ DUSP1 (MKP1) NM_004417.3 Forward 5′-CGCGCAAGTCTTCTTCCTCA-3′ Reverse 5′-GATGCTTCGCCTCTGCTTCA-3′ EDN1 NM_001955.4, NM_001168319.1 Forward 5’-TGATTTTCTCTCTGCTGTTTGTGG-3’ Reverse 5’-GCGCCTAAGACTGCTGTTTCT-3’

1 Gene Oligonucleotides Accession Number(s)

EGR1 NM_001964.2 Forward 5’-ACCTGACCGCAGAGTCTTTT-3’ Reverse 5’-GAGTGGTTTGGCTGGGGTAA-3’ EPHA4 NM_001304537.1, NM_001304536.1, NM_004438.4, XM_005246374.2 Forward 5’-GGATCATTGGAGATGGGGCT-3’ Reverse 5’-GTATTTACTCCGTCTCCGGCT-3’ FGFR2 NM_000141.4, NM_022970.3, NM_001144913.1, NM_023029.2 Forward 5’-CATCGCATTGGAGGCTACAAG-3’ NM_001144914.1, NM_001144915.1, NM_001144916.1, Reverse 5’-GGGACCACACTTTCCATAATGAG-3’ NM_001144917.1, NM_001144918.1, NM_001144919.1. GAPDH NM_002046.5, NM_001256799.2 Forward 5’-ATGGAAATCCCATCACCATCTT-3’ Reverse 5’-CAGCATCGCCCCACTTG-3’ GAS1 NM_002048.2 Forward 5’-CAGATTGTCGCCAGTGAGGA-3’ Reverse 5’-TCTGGGCCGCAGATACAAACA-3’ IL6 NM_000600.4, NM_001318095.1 Forward 5’-GAGTAGTGAGGAACAAGCCAG-3’ Reverse 5’-GTCAGGGGTGGTTATTGCATC-3’ IL11 NM_000641.3, NM_001267718.1 Forward 5’-GGGACCACAACCTGGATTC-3’ Reverse 5’-CGCAGGTAGGACAGTAGGT-3’ NR4A1 NM_002135.4, NM_173157.2, NM_001202233.1, NM_001202234.1 Forward 5’-GCATGGTGAAGGAAGTTGTCCG-3’ Reverse 5’-GGGGCTGCTTGGGTTTTGAA-3’ NR4A2 NM_006186.3 Forward 5’-GGCCCATGTCGACTCCAA-3’ Reverse 5’-GTCAGGGTTCGCCTGGAA-3’ NR4A3 NM_006981.3, NM_173200.2, NM_173199.2 Forward 5’-GTAGACAAGAGACGTCGAAACC-3’ Reverse 5’-CCTCTCCTCCCTTTCAGACTAT-3’ PDE4D NM_006203.4, NM_001104631.1, NM_001165899.1, NM_001197218.1, Forward 5’-CTCTCTGAAATGAGTCGGTCTG-3’ NM_001197219.1, NM_001197220.1, NM_001197221.1, NM_001197222.1, Reverse 5’-CCTTCTGAGTTGGAGAAGGAAT-3’ NM_001197223.1

Gene Oligonucleotides Accession Number(s) PDK4 NM_002612.3 Forward 5’-GCTGTCCATGAAGCAGCTACTG-3’ Reverse 5’-CGCAAAAATGCAAAAGAAGTTCT-3’ PPP1R3C NM_005398.6 Forward 5’-CCTAATGAGCTGCACCAGAATG-3’ Reverse 5’-TCACAGGTGGTGAATGTGCC-3’ RGS2 NM_002923 RGS2Forward 5’-CCTCAAAAGCAAGGAAAATATATACTGA-3’ Reverse 5’-AGTTGTAAAGCAGCCACTTGTAGCT-3’

RIMKLB NM_001352269.1, NM_001352270.1, NM_001352267.1, NM_001352271, Forward 5’-GCGGGGTCACAGAGGTAAAG-3’ NM_020734.4, NM_001352268.1, NM_001297776.1, XM_017019690.1, Reverse 5’-GGAACAGGTATGGCGCTTCA-3’ XM_017019683.1, XM_017019684.1, XM_017019685.1, XM_017019686.1,XM_01701968 6.1, XM_017019687.1, XM_017019688.1 SGK1 NM_001291995.1, NM_001143676.1, NM_001143677.1, NM_001143678.1 Forward 5’-ATGGCCTGCCGCCTTTTTAT-3’ NM_005627.3, XM_011536071.1 Reverse 5’-CAGGAGGTGTCTTGCGGAAT-3’ SLC7A2 NM_001164771.1, NM_001008539.3, NM_003046.5, XM_017013747, Forward 5’-GGATTCCTAGCTTTCCTCGTG-3’ XM_005273610.4, XM_005273611.4, XM_005273612.4 Reverse 5’-GCCTGGTGATGGCATGAACT-3’ SMOC1 NM_022137.5, NM_001034852.2, XM_005267995.1, XM_005267996.1 Forward 5’-GGCGTTTCACCGACTACTGT-3’ Reverse 5’-AGGCGTCCTACTTCTTTGCT-3’ SOCS3 NM_003955.3 Forward 5’-GATTCTCCTTCAATTCCTCAGCTT-3’ Reverse 5’-ATTAGTTCAGCATTCCCGAAGTGT-3’ STEAP4 NM_001205316.1, NM_001205315.1, NM_024636.3 Forward 5’-GTTCCGATTTGTCCAGTCCA-3’ Reverse 5’-GGCTGAGGAATCTCTTCCCAC-3’ TXNIP NM_006472.5, XM_017000085.1 Forward 5’-AGACCAGCCAACAGGTGAGA-3’ Reverse 5’-AGGAAGCTCAAAGCCGAACT-3’

3 Supplemental TABLE 3. Indacaterol-induced transcripts with expression level changes >1.5-fold (FDR < 0.10) at 1h, 2h, 6h and 18h.

Transcriptional Regulators Transporters, Ion Channels and Membrane Receptors Metabolic Proteins General Signalling Molecules, including Translational Regulators Other Functions Not Assigned Indacaterol (1h) (173 Probe Sets ≡ 134 Gene IDs) Affy Probe Set ID* Entrez Gene ID Official Gene Symbol FDR P-Value Fold change UniGene Name Function 204621_s_at 4929 NR4A2 2.10E-03 25.34 nuclear receptor subfamily 4, group A, member 2 207978_s_at 8013 NR4A3 2.40E-03 21.95 nuclear receptor subfamily 4, group A, member 3 216248_s_at 4929 NR4A2 1.40E-03 21.76 nuclear receptor subfamily 4, group A, member 2 204622_x_at 4929 NR4A2 1.60E-03 20.56 nuclear receptor subfamily 4, group A, member 2 209959_at 8013 NR4A3 2.80E-03 18.20 nuclear receptor subfamily 4, group A, member 3 205207_at 3569 IL6 5.78E-05 11.20 interleukin 6 205476_at 6364 CCL20 1.93E-02 7.04 chemokine (C-C motif) ligand 20 239145_at 84330 ZNF414 1.00E-01 6.62 zinc finger protein 414 228964_at 639 PRDM1 5.78E-05 6.48 PR domain containing 1, with ZNF domain 150094 SIK1 salt-inducible kinase 1 208078_s_at 2.00E-04 5.07 102724428 LOC102724428 serine/threonine-protein kinase SIK1 209774_x_at 2920 CXCL2 5.78E-05 4.91 chemokine (C-X-C motif) ligand 2 233972_s_at 55079 FEZF2 8.52E-02 4.90 FEZ family zinc finger 2 239818_x_at 10221 TRIB1 5.00E-04 4.71 tribbles pseudokinase 1 150094 SIK1 salt-inducible kinase 1 232470_at 3.00E-04 4.65 102724428 LOC102724428 serine/threonine-protein kinase SIK1 238378_at 285513 GPRIN3 1.10E-02 4.48 GPRIN family member 3 202241_at 10221 TRIB1 5.78E-05 4.46 tribbles pseudokinase 1 201739_at 6446 SGK1 1.00E-04 4.42 serum/glucocorticoid regulated kinase 1 205960_at 5166 PDK4 1.00E-04 4.41 pyruvate dehydrogenase kinase, isozyme 4 202388_at 5997 RGS2 6.58E-05 4.33 regulator of G-protein signaling 2 1557285_at 374 AREG 1.88E-02 3.79 amphiregulin 225207_at 5166 PDK4 2.90E-03 3.78 pyruvate dehydrogenase kinase, isozyme 4 218995_s_at 1906 EDN1 2.00E-04 3.63 endothelin 1 201044_x_at 1843 DUSP1 2.00E-04 3.49 dual specificity phosphatase 1 222802_at 1906 EDN1 2.00E-04 3.47 endothelin 1 1555585_a_at 153745 FAM71B 8.10E-03 3.42 family with sequence similarity 71, member B 227613_at 55422 ZNF331 2.60E-03 3.39 zinc finger protein 331 201169_s_at 8553 BHLHE40 2.00E-04 3.37 basic helix-loop-helix family, member e40 202340_x_at 3164 NR4A1 3.40E-03 3.35 nuclear receptor subfamily 4, group A, member 1 211143_x_at 3164 NR4A1 1.30E-03 3.28 nuclear receptor subfamily 4, group A, member 1 213139_at 6591 SNAI2 6.58E-05 3.24 snail family zinc finger 2 219228_at 55422 ZNF331 3.40E-03 3.22 zinc finger protein 331 244025_at 85450 ITPRIP 6.00E-04 3.17 inositol 1,4,5-trisphosphate receptor interacting protein 201170_s_at 8553 BHLHE40 7.07E-05 3.07 basic helix-loop-helix family, member e40 224797_at 57561 ARRDC3 9.10E-03 2.88 arrestin domain containing 3 205764_at 1452 CSNK1A1 2.51E-02 2.78 casein kinase 1, alpha 1 203395_s_at 3280 HES1 1.40E-02 2.77 hes family bHLH transcription factor 1 221841_s_at 9314 KLF4 3.50E-03 2.75 Kruppel-like factor 4 (gut) 225582_at 85450 ITPRIP 3.00E-04 2.66 inositol 1,4,5-trisphosphate receptor interacting protein 203394_s_at 3280 HES1 4.70E-03 2.65 hes family bHLH transcription factor 1 239530_at 119 ADD2 6.44E-02 2.64 adducin 2 (beta) 238987_at 2683 B4GALT1 1.16E-02 2.48 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1 205239_at 374 AREG 1.76E-02 2.41 amphiregulin 240413_at 149628 PYHIN1 2.32E-02 2.38 pyrin and HIN domain family, member 1 203313_s_at 7050 TGIF1 7.00E-04 2.37 TGFB-induced factor homeobox 1 210632_s_at 6442 SGCA 9.68E-02 2.35 sarcoglycan alpha 227337_at 353322 ANKRD37 7.00E-03 2.34 ankyrin repeat domain 37 220088_at 728 C5AR1 2.60E-03 2.30 complement component 5a receptor 1 36711_at 23764 MAFF 4.70E-03 2.29 v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog F 205290_s_at 650 BMP2 7.04E-02 2.27 bone morphogenetic protein 2 1559987_at 124540 MSI2 8.27E-02 2.27 musashi RNA binding protein 2 201939_at 10769 PLK2 2.60E-03 2.23 polo-like kinase 2 209582_s_at 4345 CD200 6.90E-03 2.21 CD200 molecule 1566901_at 7050 TGIF1 3.12E-02 2.21 TGFB induced factor homeobox 1 202935_s_at 6662 SOX9 9.00E-04 2.19 SRY box 9 242543_at 284948 SH2D6 8.83E-02 2.19 SH2 domain containing 6 205193_at 23764 MAFF 1.49E-02 2.17 v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog F 229152_at 260436 FDCSP 4.70E-02 2.17 follicular dendritic cell secreted protein 1555118_at 956 ENTPD3 7.24E-02 2.15 ectonucleoside triphosphate diphosphohydrolase 3 209583_s_at 4345 CD200 5.00E-04 2.12 CD200 molecule 204011_at 10253 SPRY2 8.00E-04 2.11 sprouty RTK signaling antagonist 2 204491_at 5144 PDE4D 1.67E-02 2.11 phosphodiesterase 4D, cAMP-specific 239367_at 627 BDNF 1.30E-03 2.10 brain-derived neurotrophic factor 220125_at 27019 DNAI1 5.65E-02 2.10 dynein, axonemal, intermediate chain 1 228962_at 5144 PDE4D 2.49E-02 2.06 phosphodiesterase 4D, cAMP-specific 204456_s_at 2619 GAS1 3.50E-02 2.05 growth arrest-specific 1 205466_s_at 9957 HS3ST1 2.92E-02 2.04 heparan sulfate (glucosamine) 3-O-sulfotransferase 1 203574_at 4783 NFIL3 1.33E-02 2.02 nuclear factor, interleukin 3 regulated 218177_at 57132 CHMP1B 1.63E-02 2.02 charged multivesicular body protein 1B 209457_at 1847 DUSP5 2.00E-03 2.01 dual specificity phosphatase 5 216604_s_at 23428 SLC7A8 6.90E-03 2.01 solute carrier family 7 (amino acid transporter light chain, L system), member 8 212977_at 57007 ACKR3 2.20E-03 1.99 atypical chemokine receptor 3 205027_s_at 1326 MAP3K8 2.29E-02 1.99 mitogen-activated protein kinase kinase kinase 8 206758_at 1907 EDN2 1.88E-02 1.98 endothelin 2 206382_s_at 627 BDNF 3.60E-03 1.97 brain-derived neurotrophic factor 230748_at 9120 SLC16A6 2.20E-02 1.96 solute carrier family 16, member 6 207826_s_at 3399 ID3 5.10E-03 1.95 inhibitor of DNA binding 3, dominant negative helix-loop-helix protein 206706_at 4908 NTF3 8.00E-04 1.94 neurotrophin 3 218631_at 60370 AVPI1 9.00E-04 1.93 arginine vasopressin-induced 1 218178_s_at 57132 CHMP1B 1.80E-03 1.93 charged multivesicular body protein 1B 202936_s_at 6662 SOX9 1.90E-03 1.93 SRY box 9 1139 CHRFAM7A 3.12E-02 CHRNA7 (Exons 5-10) And FAM7A (Exons A-E) Fusion 236385_at 89832 CHRNA7 3.12E-02 1.93 cholinergic receptor, nicotinic alpha 7 101929970 LOC101929970 3.12E-02 CHRNA7-FAM7A fusion protein 229110_at 25769 SLC24A2 5.09E-02 1.93 solute carrier family 24 (sodium/potassium/calcium exchanger), member 2 202672_s_at 467 ATF3 5.76E-02 1.93 activating transcription factor 3 208937_s_at 3397 ID1 1.21E-02 1.91 inhibitor of DNA binding 1, dominant negative helix-loop-helix protein 218810_at 80149 ZC3H12A 9.20E-03 1.90 zinc finger CCCH-type containing 12A 208960_s_at 1316 KLF6 6.00E-04 1.88 Kruppel-like factor 6 202023_at 1942 EFNA1 3.00E-04 1.86 ephrin-A1 215111_s_at 8848 TSC22D1 1.03E-02 1.85 TSC22 domain family, member 1 218723_s_at 28984 RGCC 1.33E-02 1.85 regulator of cell cycle 239058_at 2303 FOXC2 1.49E-02 1.85 forkhead box C2 232746_at 57007 ACKR3 4.43E-02 1.84 atypical chemokine receptor 3 225557_at 64651 CSRNP1 2.20E-03 1.83 cysteine-serine-rich nuclear protein 1 207850_at 2921 CXCL3 9.80E-03 1.83 chemokine (C-X-C motif) ligand 3 234583_at 10752 CHL1 6.34E-02 1.83 cell adhesion molecule L1-like 1552948_at 148870 CCDC27 9.80E-03 1.82 coiled-coil domain containing 27 56672 AKIP1 2.00E-03 1.81 A kinase (PRKA) interacting protein 1 220987_s_at 81788 NUAK2 2.00E-03 1.81 NUAK family, SNF1-like kinase, 2 202149_at 4739 NEDD9 8.40E-03 1.81 neural precursor cell expressed, developmentally down-regulated 9 202431_s_at 4609 MYC 6.00E-04 1.80 v-myc avian myelocytomatosis viral oncogene homolog 220266_s_at 9314 KLF4 4.10E-02 1.80 Kruppel-like factor 4 (gut) 205815_at 5068 REG3A 8.98E-02 1.79 regenerating islet-derived 3 alpha 202499_s_at 6515 SLC2A3 3.34E-02 1.78 solute carrier family 2 (facilitated glucose transporter), member 3 204014_at 1846 DUSP4 1.20E-02 1.77 dual specificity phosphatase 4 210836_x_at 5144 PDE4D 1.90E-02 1.77 phosphodiesterase 4D, cAMP-specific 1555832_s_at 1316 KLF6 1.40E-03 1.76 Kruppel-like factor 6 203708_at 5142 PDE4B 1.96E-02 1.76 phosphodiesterase 4B, cAMP-specific 226034_at 1846 DUSP4 6.90E-03 1.75 dual specificity phosphatase 4 230472_at 79192 IRX1 6.90E-03 1.75 iroquois homeobox 1 202150_s_at 4739 NEDD9 2.18E-02 1.74 neural precursor cell expressed, developmentally down-regulated 9 231035_s_at 220213 OTUD1 3.90E-02 1.74 OTU deubiquitinase 1 211962_s_at 677 ZFP36L1 9.92E-02 1.73 ZFP36 ring finger protein-like 1 235849_at 286133 SCARA5 6.63E-02 1.71 scavenger receptor class A, member 5 1553422_s_at 54715 RBFOX1 5.93E-02 1.70 RNA binding protein, fox-1 homolog (C. elegans) 1 1553613_s_at 2296 FOXC1 1.60E-03 1.69 forkhead box C1 233379_at 79899 PRR5L 8.33E-02 1.69 proline rich 5 like 1554980_a_at 467 ATF3 1.80E-02 1.68 activating transcription factor 3 215990_s_at 604 BCL6 1.90E-02 1.68 B-cell CLL/lymphoma 6 222162_s_at 9510 ADAMTS1 2.20E-02 1.68 ADAM metallopeptidase with thrombospondin type 1 motif 1 202497_x_at 6515 SLC2A3 2.97E-02 1.68 solute carrier family 2 (facilitated glucose transporter), member 3 6515 SLC2A14 2.98E-02 solute carrier family 2 (facilitated glucose transporter), member 14 216236_s_at 144195 SLC2A3 2.98E-02 1.68 solute carrier family 2 (facilitated glucose transporter), member 3 213170_at 2882 GPX7 4.29E-02 glutathione peroxidase 7 224606_at 1316 KLF6 2.30E-03 1.67 Kruppel-like factor 6 203140_at 604 BCL6 2.60E-03 1.67 B-cell CLL/lymphoma 6 207981_s_at 2104 ESRRG 4.29E-02 1.67 estrogen-related receptor gamma 213284_at 677 ZFP36L1 5.24E-02 1.66 ZFP36 ring finger protein-like 1 224657_at 54206 ERRFI1 1.45E-02 1.65 ERBB receptor feedback inhibitor 1 204931_at 6943 TCF21 1.88E-02 1.65 transcription factor 21 204284_at 5507 PPP1R3C 5.76E-02 1.65 protein phosphatase 1, regulatory subunit 3C 201041_s_at 1843 DUSP1 6.00E-04 1.64 dual specificity phosphatase 1 221766_s_at 55603 FAM46A 5.09E-02 1.64 family with sequence similarity 46, member A 227443_at 286343 LURAP1L 7.04E-02 1.64 leucine rich adaptor protein 1-like 229373_at 3274 HRH2 1.63E-02 1.63 histamine receptor H2 6515 SLC2A14 6.27E-02 1.63 solute carrier family 2 (facilitated glucose transporter), member 14 222088_s_at 144195 SLC2A3 6.27E-02 1.63 solute carrier family 2 (facilitated glucose transporter), member 3 205929_at 10223 GPA33 6.88E-02 1.62 glycoprotein A33 (transmembrane) 1554420_at 467 ATF3 9.65E-02 1.62 activating transcription factor 3 222379_at 23704 KCNE4 1.88E-02 1.61 potassium channel, voltage gated subfamily E regulatory beta subunit 4 219371_s_at 10365 KLF2 4.72E-02 1.61 Kruppel-like factor 2 225720_at 171024 SYNPO2 8.98E-02 1.61 synaptopodin 2 208961_s_at 1316 KLF6 1.80E-03 1.60 Kruppel-like factor 6 225606_at 10018 BCL2L11 2.32E-02 1.60 BCL2-like 11 (apoptosis facilitator) 1553378_a_at 256957 HEATR9 5.39E-02 1.60 HEAT repeat containing 9 234928_x_at 864 RUNX3 6.63E-02 1.60 runt-related transcription factor 3 206374_at 1850 DUSP8 2.31E-02 1.59 dual specificity phosphatase 8 1431_at 1571 CYP2E1 1.67E-02 1.58 cytochrome P450, family 2, subfamily E, polypeptide 1 218880_at 2355 FOSL2 4.79E-02 1.58 FOS-like antigen 2 211302_s_at 5142 PDE4B 8.43E-02 1.58 phosphodiesterase 4B, cAMP-specific 239381_at 5650 KLK7 9.47E-02 1.58 kallikrein related peptidase 7 203438_at 8614 STC2 1.63E-02 1.57 stanniocalcin 2 226140_s_at 220213 OTUD1 8.90E-03 1.56 OTU deubiquitinase 1 8739 HRK 8.49E-02 harakiri, BCL2 interacting protein 229552_at 1.56 283454 LOC283454 8.49E-02 uncharacterized LOC283454 201328_at 2114 ETS2 7.00E-03 1.55 v-ets avian erythroblastosis virus E26 oncogene homolog 2 228325_at 23514 SPIDR 1.03E-02 1.55 scaffolding protein involved in DNA repair 228462_at 153572 IRX2 1.33E-02 1.55 iroquois homeobox 2 212143_s_at 3486 IGFBP3 3.90E-02 1.55 insulin like growth factor binding protein 3 228188_at 2355 FOSL2 8.83E-02 1.55 FOS-like antigen 2 230493_at 387914 SHISA2 8.30E-03 1.54 shisa family member 2 207980_s_at 10370 CITED2 1.11E-02 1.54 Cbp/p300-interacting transactivator, with Glu/Asp rich carboxy-terminal domain, 2 212230_at 8613 PLPP3 3.27E-02 1.54 phospholipid phosphatase 3 202704_at 10140 TOB1 5.18E-02 1.54 transducer of ERBB2, 1 209101_at 1490 CTGF 5.89E-02 1.54 connective tissue growth factor 202628_s_at 5054 SERPINE1 2.60E-03 1.53 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 203973_s_at 1052 CEBPD 7.00E-03 1.53 CCAAT/enhancer binding protein (C/EBP), delta 231958_at 132001 TAMM41 3.78E-02 1.53 TAM41 mitochondrial translocator assembly and maintenance homolog 228167_at 89857 KLHL6 7.24E-02 1.53 kelch-like family member 6 229800_at 9201 DCLK1 1.63E-02 1.52 doublecortin-like kinase 1 212226_s_at 8613 PLPP3 1.72E-02 1.52 phospholipid phosphatase 3 206999_at 3595 IL12RB2 4.25E-02 1.52 interleukin 12 receptor, beta 2 206170_at 154 ADRB2 1.63E-02 1.51 adrenoceptor beta 2, surface 229004_at 170689 ADAMTS15 1.88E-02 1.51 ADAM metallopeptidase with thrombospondin type 1 motif 15 230372_at 3037 HAS2 2.25E-02 1.51 hyaluronan synthase 2 204015_s_at 1846 DUSP4 2.41E-02 1.51 dual specificity phosphatase 4 230773_at 79750 ZNF385D 3.60E-02 1.51 zinc finger protein 385D 208293_x_at 1444 CSHL1 6.70E-02 1.51 chorionic somatomammotropin hormone-like 1 235182_at 140862 ISM1 9.00E-02 1.51 isthmin 1, angiogenesis inhibitor

Indacaterol (2h) (515 Probe Sets ≡ 321 Gene IDs) Affy Probe Set ID* Entrez Gene ID Official Gene Symbol FDR P-Value Fold change UniGene Name Function 209959_at 8013 NR4A3 6.05E-05 66.19 nuclear receptor subfamily 4, group A, member 3 207978_s_at 8013 NR4A3 4.57E-05 38.53 nuclear receptor subfamily 4, group A, member 3 216248_s_at 4929 NR4A2 7.00E-04 28.71 nuclear receptor subfamily 4, group A, member 2 204622_x_at 4929 NR4A2 2.00E-04 24.49 nuclear receptor subfamily 4, group A, member 2 204621_s_at 4929 NR4A2 1.70E-03 24.45 nuclear receptor subfamily 4, group A, member 2 203639_s_at 2263 FGFR2 1.51E-02 13.55 fibroblast growth factor receptor 2 205289_at 650 BMP2 1.45E-02 11.62 bone morphogenetic protein 2 221541_at 83716 CRISPLD2 5.25E-06 8.50 cysteine-rich secretory protein LCCL domain containing 2 225207_at 5166 PDK4 1.00E-04 8.30 pyruvate dehydrogenase kinase, isozyme 4 205476_at 6364 CCL20 3.00E-04 7.94 chemokine (C-C motif) ligand 20 205207_at 3569 IL6 1.90E-03 7.75 interleukin 6 234511_at 140731 ANKRD60 1.08E-01 7.53 ankyrin repeat domain 60 205960_at 5166 PDK4 3.00E-03 6.74 pyruvate dehydrogenase kinase, isozyme 4 150094 SIK1 salt-inducible kinase 1 2.00E-04 6.56 232470_at 102724428 LOC102724428 serine/threonine-protein kinase SIK1 150094 SIK1 salt-inducible kinase 1 208078_s_at 1.11E-05 6.31 102724428 LOC102724428 serine/threonine-protein kinase SIK1 218541_s_at 56892 C8orf4 1.81E-02 6.12 chromosome 8 open reading frame 4 228962_at 5144 PDE4D 8.63E-05 6.12 phosphodiesterase 4D, cAMP-specific 201739_at 6446 SGK1 6.05E-05 5.78 serum/glucocorticoid regulated kinase 1 206359_at 9021 SOCS3 1.44E-02 5.73 suppressor of cytokine signaling 3 209582_s_at 4345 CD200 2.00E-04 5.71 CD200 molecule 203638_s_at 2263 FGFR2 1.20E-03 5.67 fibroblast growth factor receptor 2 228964_at 639 PRDM1 1.11E-05 5.50 PR domain containing 1, with ZNF domain 211401_s_at 2263 FGFR2 5.23E-02 5.37 fibroblast growth factor receptor 2 204491_at 5144 PDE4D 5.15E-05 5.25 phosphodiesterase 4D, cAMP-specific 227697_at 9021 SOCS3 2.11E-02 5.04 suppressor of cytokine signaling 3 225582_at 85450 ITPRIP 1.35E-05 4.75 inositol 1,4,5-trisphosphate receptor interacting protein 210837_s_at 5144 PDE4D 2.00E-04 4.75 phosphodiesterase 4D, cAMP-specific 202388_at 5997 RGS2 9.78E-05 4.75 regulator of G-protein signaling 2 210836_x_at 5144 PDE4D 9.33E-05 4.52 phosphodiesterase 4D, cAMP-specific 244025_at 85450 ITPRIP 3.00E-04 4.49 inositol 1,4,5-trisphosphate receptor interacting protein 205290_s_at 650 BMP2 1.20E-03 4.45 bone morphogenetic protein 2 209774_x_at 2920 CXCL2 1.80E-03 4.40 chemokine (C-X-C motif) ligand 2 205330_at 4330 MN1 1.20E-03 4.40 meningioma (disrupted in balanced translocation) 1 1562321_at 5166 PDK4 1.34E-01 4.35 pyruvate dehydrogenase kinase, isozyme 4 230748_at 9120 SLC16A6 1.00E-04 4.32 solute carrier family 16, member 6 209583_s_at 4345 CD200 1.11E-05 4.30 CD200 molecule 227613_at 55422 ZNF331 2.00E-04 4.30 zinc finger protein 331 207038_at 9120 SLC16A6 3.20E-03 4.29 solute carrier family 16, member 6 220088_at 728 C5AR1 5.00E-04 4.23 complement component 5a receptor 1 211143_x_at 3164 NR4A1 5.00E-04 4.17 nuclear receptor subfamily 4, group A, member 1 219228_at 55422 ZNF331 3.00E-04 4.12 zinc finger protein 331 8739 HRK harakiri, BCL2 interacting protein 229552_at 3.00E-04 4.08 283454 LOC283454 uncharacterized LOC283454 239367_at 627 BDNF 3.74E-05 4.05 brain-derived neurotrophic factor 3694 ITGB6 integrin beta 6 208083_s_at 7.49E-02 4.05 100505984 LOC100505984 uncharacterized LOC100505984 213182_x_at 1028 CDKN1C 2.68E-02 4.02 cyclin-dependent kinase inhibitor 1C (p57, Kip2) 202340_x_at 3164 NR4A1 1.60E-03 3.97 nuclear receptor subfamily 4, group A, member 1 219911_s_at 28231 SLCO4A1 2.40E-03 3.84 solute carrier organic anion transporter family, member 4A1 157693 FAM87A family with sequence similarity 87, member A 1559141_s_at 8.00E-03 3.81 400728 FAM87B family with sequence similarity 87, member B 219534_x_at 1028 CDKN1C 1.81E-02 3.76 cyclin-dependent kinase inhibitor 1C (p57, Kip2) 224828_at 80315 CPEB4 3.00E-04 3.74 cytoplasmic polyadenylation element binding protein 4 229709_at 483 ATP1B3 6.20E-03 3.73 ATPase, Na+/K+ transporting, beta 3 polypeptide 206382_s_at 627 BDNF 1.00E-04 3.72 brain-derived neurotrophic factor 219682_s_at 6926 TBX3 9.99E-05 3.59 T-box 3 203549_s_at 4023 LPL 2.00E-04 3.57 lipoprotein lipase 224829_at 80315 CPEB4 2.00E-04 3.53 cytoplasmic polyadenylation element binding protein 4 216894_x_at 1028 CDKN1C 7.80E-03 3.47 cyclin-dependent kinase inhibitor 1C (p57, Kip2) 206865_at 8739 HRK 2.00E-04 3.47 harakiri, BCL2 interacting protein 239818_x_at 10221 TRIB1 2.00E-04 3.46 tribbles pseudokinase 1 218177_at 57132 CHMP1B 2.00E-04 3.39 charged multivesicular body protein 1B 218995_s_at 1906 EDN1 8.59E-05 3.36 endothelin 1 1552755_at 157983 C9orf66 8.11E-02 3.35 chromosome 9 open reading frame 66 6515 SLC2A14 solute carrier family 2 (facilitated glucose transporter), member 14 216236_s_at 9.78E-05 3.35 144195 SLC2A3 solute carrier family 2 (facilitated glucose transporter), member 3 213348_at 1028 CDKN1C 8.70E-03 3.32 cyclin-dependent kinase inhibitor 1C (p57, Kip2) 213006_at 1052 CEBPD 8.00E-04 3.32 CCAAT/enhancer binding protein (C/EBP), delta 202241_at 10221 TRIB1 3.74E-05 3.31 tribbles pseudokinase 1 209631_s_at 2861 GPR37 2.00E-04 3.30 G protein-coupled receptor 37 (endothelin receptor type B-like) 202497_x_at 6515 SLC2A3 2.00E-04 3.30 solute carrier family 2 (facilitated glucose transporter), member 3 228128_x_at 5069 PAPPA 4.00E-04 3.28 pregnancy-associated plasma protein A, pappalysin 1 201981_at 5069 PAPPA 2.68E-02 3.21 pregnancy-associated plasma protein A, pappalysin 1 203548_s_at 4023 LPL 2.00E-04 3.18 lipoprotein lipase 224831_at 80315 CPEB4 2.00E-04 3.17 cytoplasmic polyadenylation element binding protein 4 6515 SLC2A14 solute carrier family 2 (facilitated glucose transporter), member 14 222088_s_at 3.00E-04 3.16 144195 SLC2A3 solute carrier family 2 (facilitated glucose transporter), member 3 228325_at 23514 SPIDR 8.00E-04 3.16 scaffolding protein involved in DNA repair 220483_s_at 25897 RNF19A 5.00E-04 3.13 ring finger protein 19A, RBR E3 ubiquitin protein ligase 202499_s_at 6515 SLC2A3 5.00E-04 3.11 solute carrier family 2 (facilitated glucose transporter), member 3 224942_at 5069 PAPPA 7.00E-04 3.09 pregnancy-associated plasma protein A, pappalysin 1 202498_s_at 6515 SLC2A3 1.00E-03 3.04 solute carrier family 2 (facilitated glucose transporter), member 3 224940_s_at 5069 PAPPA 8.00E-04 3.03 pregnancy-associated plasma protein A, pappalysin 1 239123_at 8848 TSC22D1 1.21E-02 3.03 TSC22 domain family, member 1 218178_s_at 57132 CHMP1B 1.77E-05 3.00 charged multivesicular body protein 1B 203984_s_at 842 CASP9 4.80E-03 2.99 caspase 9 222802_at 1906 EDN1 8.34E-05 2.98 endothelin 1 226614_s_at 83648 FAM167A 5.00E-04 2.96 family with sequence similarity 167, member A 205027_s_at 1326 MAP3K8 1.70E-03 2.93 mitogen-activated protein kinase kinase kinase 8 204456_s_at 2619 GAS1 5.50E-03 2.91 growth arrest-specific 1 212226_s_at 8613 PLPP3 6.82E-05 2.90 phospholipid phosphatase 3 218723_s_at 28984 RGCC 1.02E-02 2.89 regulator of cell cycle 212977_at 57007 ACKR3 9.84E-05 2.87 atypical chemokine receptor 3 208228_s_at 2263 FGFR2 1.00E-04 2.86 fibroblast growth factor receptor 2 225516_at 6542 SLC7A2 1.00E-04 2.84 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 209355_s_at 8613 PLPP3 7.59E-05 2.83 phospholipid phosphatase 3 225544_at 6926 TBX3 1.00E-04 2.82 T-box 3 230472_at 79192 IRX1 7.00E-04 2.81 iroquois homeobox 1 210775_x_at 842 CASP9 5.20E-03 2.77 caspase 9 237187_at 8739 HRK 3.20E-03 2.77 harakiri, BCL2 interacting protein 224941_at 5069 PAPPA 2.60E-03 2.77 pregnancy-associated plasma protein A, pappalysin 1 225606_at 10018 BCL2L11 1.00E-04 2.76 BCL2-like 11 (apoptosis facilitator) 219316_s_at 55640 FLVCR2 2.20E-03 2.72 feline leukemia virus subgroup C cellular receptor family, member 2 228340_at 7090 TLE3 5.60E-02 2.69 transducin-like enhancer of split 3 1553613_s_at 2296 FOXC1 1.54E-05 2.67 forkhead box C1 209681_at 10560 SLC19A2 4.50E-03 2.67 solute carrier family 19 (thiamine transporter), member 2 235085_at 157285 PRAG1 5.20E-03 2.65 homolog of rat pragma of Rnd2 214446_at 22936 ELL2 1.02E-02 2.63 elongation factor, RNA polymerase II, 2 209189_at 2353 FOS 6.00E-03 2.63 FBJ murine osteosarcoma viral oncogene homolog 223085_at 25897 RNF19A 2.00E-04 2.62 ring finger protein 19A, RBR E3 ubiquitin protein ligase 1454 CSNK1E casein kinase 1, epsilon 222015_at 5.00E-04 2.61 102800317 LOC400927-CSNK1E LOC400927-CSNK1E readthrough 227443_at 286343 LURAP1L 1.36E-02 2.61 leucine rich adaptor protein 1-like 205239_at 374 AREG 4.20E-03 2.60 amphiregulin 218880_at 2355 FOSL2 3.00E-04 2.60 FOS-like antigen 2 224325_at 8325 FZD8 9.00E-03 2.60 frizzled class receptor 8 227099_s_at 387763 C11orf96 6.76E-02 2.59 chromosome 11 open reading frame 96 227188_at 59271 EVA1C 8.50E-03 2.59 eva-1 homolog C (C. elegans) 206924_at 3589 IL11 3.00E-03 2.59 interleukin 11 229004_at 170689 ADAMTS15 9.99E-05 2.58 ADAM metallopeptidase with thrombospondin type 1 motif 15 214438_at 3142 HLX 3.38E-02 2.57 H2.0-like homeobox 36711_at 23764 MAFF 1.00E-04 2.57 v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog F 205193_at 23764 MAFF 7.00E-04 2.57 v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog F 227337_at 353322 ANKRD37 3.00E-04 2.56 ankyrin repeat domain 37 220225_at 50805 IRX4 6.60E-03 2.56 iroquois homeobox 4 218631_at 60370 AVPI1 6.05E-05 2.55 arginine vasopressin-induced 1 225987_at 79689 STEAP4 4.00E-04 2.54 STEAP family member 4 230398_at 84951 TNS4 1.11E-02 2.54 tensin 4 215111_s_at 8848 TSC22D1 4.60E-03 2.53 TSC22 domain family, member 1 205409_at 2355 FOSL2 5.70E-03 2.52 FOS-like antigen 2 1553133_at 203228 C9orf72 2.60E-03 2.51 chromosome 9 open reading frame 72 212230_at 8613 PLPP3 3.26E-05 2.51 phospholipid phosphatase 3 201044_x_at 1843 DUSP1 2.00E-04 2.50 dual specificity phosphatase 1 208250_s_at 1755 DMBT1 8.63E-05 2.48 deleted in malignant brain tumors 1 236911_at 57494 RIMKLB 2.53E-02 2.48 ribosomal modification protein rimK-like family member B 202150_s_at 4739 NEDD9 4.00E-04 2.47 neural precursor cell expressed, developmentally down-regulated 9 202723_s_at 2308 FOXO1 3.20E-03 2.46 forkhead box O1 202768_at 2354 FOSB 7.40E-03 2.43 FBJ murine osteosarcoma viral oncogene homolog B 206404_at 2254 FGF9 4.00E-04 2.42 fibroblast growth factor 9 213221_s_at 23235 SIK2 3.30E-03 2.42 salt-inducible kinase 2 205466_s_at 9957 HS3ST1 2.21E-02 2.40 heparan sulfate (glucosamine) 3-O-sulfotransferase 1 201169_s_at 8553 BHLHE40 5.60E-03 2.39 basic helix-loop-helix family, member e40 210136_at 4155 MBP 7.00E-04 2.39 myelin basic protein 202149_at 4739 NEDD9 3.00E-04 2.38 neural precursor cell expressed, developmentally down-regulated 9 203574_at 4783 NFIL3 5.00E-04 2.38 nuclear factor, interleukin 3 regulated 223379_s_at 26524 LATS2 3.00E-04 2.37 large tumor suppressor kinase 2 207626_s_at 6542 SLC7A2 3.00E-04 2.37 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 213139_at 6591 SNAI2 5.20E-03 2.37 snail family zinc finger 2 224797_at 57561 ARRDC3 5.80E-03 2.36 arrestin domain containing 3 215990_s_at 604 BCL6 9.30E-03 2.36 B-cell CLL/lymphoma 6 224480_s_at 84803 GPAT3 1.27E-01 2.36 glycerol-3-phosphate acyltransferase 3 231035_s_at 220213 OTUD1 3.70E-03 2.36 OTU deubiquitinase 1 1454 CSNK1E casein kinase 1, epsilon 226858_at 4.00E-04 2.34 102800317 LOC400927-CSNK1E LOC400927-CSNK1E readthrough 209101_at 1490 CTGF 3.00E-04 2.34 connective tissue growth factor 231067_s_at 9590 AKAP12 1.51E-02 2.33 A kinase (PRKA) anchor protein 12 220335_x_at 23491 CES3 1.70E-03 2.33 carboxylesterase 3 216627_s_at 2683 B4GALT1 6.80E-03 2.32 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1 218881_s_at 2355 FOSL2 3.00E-03 2.32 FOS-like antigen 2 237252_at 7056 THBD 1.58E-02 2.32 thrombomodulin 208606_s_at 54361 WNT4 9.27E-02 2.32 wingless-type MMTV integration site family, member 4 208536_s_at 10018 BCL2L11 2.11E-02 2.28 BCL2-like 11 (apoptosis facilitator) 203796_s_at 605 BCL7A 4.00E-04 2.28 B-cell CLL/lymphoma 7A 212099_at 388 RHOB 4.00E-04 2.28 ras homolog family member B 203313_s_at 7050 TGIF1 6.00E-04 2.28 TGFB-induced factor homeobox 1 223484_at 84419 C15orf48 1.32E-01 2.27 chromosome 15 open reading frame 48 229720_at 573 BAG1 1.50E-03 2.25 BCL2-associated athanogene 1558143_a_at 10018 BCL2L11 5.00E-03 2.25 BCL2-like 11 (apoptosis facilitator) 219195_at 10891 PPARGC1A 1.14E-02 2.24 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha 223430_at 23235 SIK2 1.84E-02 2.23 salt-inducible kinase 2 207213_s_at 9099 USP2 3.00E-03 2.23 ubiquitin specific peptidase 2 238987_at 2683 B4GALT1 8.84E-02 2.22 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1 228625_at 163732 CITED4 4.00E-04 2.22 Cbp/p300-interacting transactivator, with Glu/Asp rich carboxy-terminal domain, 4 225955_at 284207 METRNL 1.00E-04 2.22 meteorin, glial cell differentiation regulator-like 1554544_a_at 4155 MBP 7.00E-04 2.21 myelin basic protein 200731_s_at 7803 PTP4A1 1.09E-02 2.21 protein tyrosine phosphatase type IVA, member 1 203887_s_at 7056 THBD 1.69E-02 2.21 thrombomodulin 202023_at 1942 EFNA1 2.00E-04 2.20 ephrin-A1 221841_s_at 9314 KLF4 9.90E-03 2.20 Kruppel-like factor 4 (gut) 206472_s_at 7090 TLE3 8.63E-02 2.19 transducin-like enhancer of split 3 201170_s_at 8553 BHLHE40 4.00E-04 2.18 basic helix-loop-helix family, member e40 202672_s_at 467 ATF3 8.00E-04 2.17 activating transcription factor 3 228188_at 2355 FOSL2 8.00E-04 2.17 FOS-like antigen 2 221011_s_at 81606 LBH 3.00E-04 2.17 limb bud and heart development 222784_at 64093 SMOC1 3.10E-02 2.17 SPARC related modular calcium binding 1 226575_at 58499 ZNF462 1.00E-03 2.17 zinc finger protein 462 219433_at 54880 BCOR 1.35E-02 2.16 BCL6 corepressor 205870_at 624 BDKRB2 3.27E-02 2.16 bradykinin receptor B2 204014_at 1846 DUSP4 5.20E-03 2.16 dual specificity phosphatase 4 225262_at 2355 FOSL2 1.00E-04 2.16 FOS-like antigen 2 209184_s_at 8660 IRS2 7.00E-04 2.15 insulin receptor substrate 2 213260_at 2296 FOXC1 5.34E-05 2.14 forkhead box C1 234804_at 283571 PROX2 1.29E-01 2.14 prospero homeobox 2 200732_s_at 7803 PTP4A1 2.30E-03 2.14 protein tyrosine phosphatase type IVA, member 1 206035_at 5966 REL 2.90E-03 2.13 v-rel avian reticuloendotheliosis viral oncogene homolog 205896_at 6583 SLC22A4 2.08E-02 2.13 solute carrier family 22 (organic cation/zwitterion transporter), member 4 203290_at 3117 HLA-DQA1 3.73E-02 2.12 major histocompatibility complex, class II, DQ alpha 1 1553962_s_at 388 RHOB 1.40E-03 2.12 ras homolog family member B 1556185_a_at 79689 STEAP4 3.99E-02 2.12 STEAP family member 4 212143_s_at 3486 IGFBP3 1.70E-03 2.11 insulin like growth factor binding protein 3 204032_at 8412 BCAR3 4.00E-03 2.10 breast cancer anti-estrogen resistance 3 211631_x_at 2683 B4GALT1 3.66E-02 2.09 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1 209772_s_at 100133941 CD24 3.00E-04 2.09 CD24 molecule 224572_s_at 359948 IRF2BP2 3.00E-04 2.09 interferon regulatory factor 2 binding protein 2 233002_at 57718 PPP4R4 1.53E-02 2.09 protein phosphatase 4, regulatory subunit 4 207826_s_at 3399 ID3 1.10E-03 2.08 inhibitor of DNA binding 3, dominant negative helix-loop-helix protein 233379_at 79899 PRR5L 1.35E-02 2.08 proline rich 5 like 225999_at 57494 RIMKLB 3.10E-03 2.08 ribosomal modification protein rimK-like family member B 203140_at 604 BCL6 9.00E-04 2.07 B-cell CLL/lymphoma 6 223566_s_at 54880 BCOR 5.30E-03 2.07 BCL6 corepressor 227458_at 29126 CD274 3.20E-03 2.07 CD274 molecule 202724_s_at 2308 FOXO1 1.80E-03 2.07 forkhead box O1 205051_s_at 3815 KIT 2.70E-03 2.07 v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog 205157_s_at 3872 KRT17 3.12E-02 2.07 keratin 17, type I 225407_at 4155 MBP 8.00E-04 2.07 myelin basic protein 223834_at 29126 CD274 5.60E-03 2.06 CD274 molecule 209457_at 1847 DUSP5 1.90E-03 2.06 dual specificity phosphatase 5 230348_at 26524 LATS2 7.70E-03 2.06 large tumor suppressor kinase 2 204284_at 5507 PPP1R3C 2.53E-02 2.06 protein phosphatase 1, regulatory subunit 3C 218113_at 23670 TMEM2 7.00E-04 2.06 transmembrane protein 2 227529_s_at 9590 AKAP12 9.90E-03 2.05 A kinase (PRKA) anchor protein 12 203795_s_at 605 BCL7A 3.50E-03 2.05 B-cell CLL/lymphoma 7A 219383_at 79899 PRR5L 1.70E-03 2.05 proline rich 5 like 223916_s_at 54880 BCOR 3.60E-02 2.04 BCL6 corepressor 216598_s_at 6347 CCL2 2.10E-03 2.03 chemokine (C-C motif) ligand 2 201925_s_at 1604 CD55 1.99E-02 2.03 CD55 molecule, decay accelerating factor for complement (Cromer blood group) 228462_at 153572 IRX2 6.00E-04 2.03 iroquois homeobox 2 220540_at 60598 KCNK15 7.93E-02 2.03 potassium channel, two pore domain subfamily K, member 15 231798_at 9241 NOG 6.00E-04 2.03 noggin 217584_at 4864 NPC1 2.11E-02 2.03 Niemann-Pick disease, type C1 228284_at 7088 TLE1 4.00E-03 2.03 transducin-like enhancer of split 1 (E(sp1) homolog, Drosophila) 241547_at 29974 A1CF 4.11E-02 2.02 APOBEC1 complementation factor 203708_at 5142 PDE4B 2.22E-02 2.02 phosphodiesterase 4B, cAMP-specific 225919_s_at 203228 C9orf72 3.44E-02 2.01 chromosome 9 open reading frame 72 225626_at 55824 PAG1 5.80E-02 2.01 phosphoprotein membrane anchor with glycosphingolipid microdomains 1 1554980_a_at 467 ATF3 8.00E-04 2.00 activating transcription factor 3 1555950_a_at 1604 CD55 6.90E-03 2.00 CD55 molecule, decay accelerating factor for complement (Cromer blood group) 213844_at 3202 HOXA5 9.00E-03 2.00 homeobox A5 206864_s_at 8739 HRK 5.00E-04 2.00 harakiri, BCL2 interacting protein 1555486_a_at 79899 PRR5L 2.00E-04 2.00 proline rich 5 like 204654_s_at 7020 TFAP2A 6.70E-03 2.00 transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha) 239178_at 2254 FGF9 2.11E-02 1.99 fibroblast growth factor 9 203108_at 9052 GPRC5A 6.00E-04 1.99 G protein-coupled receptor, class C, group 5, member A 220187_at 79689 STEAP4 9.40E-03 1.99 STEAP family member 4 229337_at 9099 USP2 2.33E-02 1.99 ubiquitin specific peptidase 2 236352_at 245806 VGLL2 1.53E-02 1.99 vestigial-like family member 2 242162_at 164781 DAW1 1.51E-02 1.98 dynein assembly factor with WDR repeat domains 1 226206_at 7975 MAFK 3.00E-04 1.98 v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog K 235421_at 1326 MAP3K8 4.99E-02 1.98 mitogen-activated protein kinase kinase kinase 8 224570_s_at 359948 IRF2BP2 3.20E-03 1.97 interferon regulatory factor 2 binding protein 2 229889_at 388341 LRRC75A 1.78E-02 1.97 leucine rich repeat containing 75A 228293_at 91614 DEPDC7 1.70E-03 1.96 DEP domain containing 7 201328_at 2114 ETS2 7.00E-04 1.96 v-ets avian erythroblastosis virus E26 oncogene homolog 2 210162_s_at 4772 NFATC1 8.00E-04 1.96 nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 219737_s_at 5101 PCDH9 6.30E-03 1.96 protocadherin 9 218691_s_at 8572 PDLIM4 1.00E-01 1.96 PDZ and LIM domain 4 212444_at 9052 GPRC5A 8.00E-04 1.95 G protein-coupled receptor, class C, group 5, member A 228812_at 5966 REL 6.90E-03 1.95 v-rel avian reticuloendotheliosis viral oncogene homolog 222343_at 10018 BCL2L11 5.00E-03 1.94 BCL2-like 11 (apoptosis facilitator) 224826_at 56261 GPCPD1 2.40E-03 1.94 glycerophosphocholine phosphodiesterase 1 203372_s_at 8835 SOCS2 6.82E-02 1.94 suppressor of cytokine signaling 2 226034_at 1846 DUSP4 1.60E-03 1.93 dual specificity phosphatase 4 224835_at 56261 GPCPD1 4.20E-03 1.93 glycerophosphocholine phosphodiesterase 1 201473_at 3726 JUNB 1.15E-01 1.93 jun B proto-oncogene 215034_s_at 4071 TM4SF1 2.90E-02 1.93 transmembrane 4 L six family member 1 227530_at 9590 AKAP12 1.16E-02 1.92 A kinase (PRKA) anchor protein 12 208650_s_at 100133941 CD24 4.10E-03 1.92 CD24 molecule 225408_at 4155 MBP 1.00E-03 1.92 myelin basic protein 206036_s_at 5966 REL 6.80E-03 1.92 v-rel avian reticuloendotheliosis viral oncogene homolog 205074_at 6584 SLC22A5 4.20E-03 1.92 solute carrier family 22 (organic cation/carnitine transporter), member 5 231183_s_at 128710 SLX4IP 3.54E-02 1.92 SLX4 interacting protein 205286_at 7022 TFAP2C 3.09E-02 1.92 transcription factor AP-2 gamma (activating enhancer binding protein 2 gamma) 209185_s_at 8660 IRS2 2.00E-04 1.91 insulin receptor substrate 2 238419_at 90102 PHLDB2 8.17E-02 1.91 pleckstrin homology-like domain, family B, member 2 210095_s_at 3486 IGFBP3 9.00E-04 1.90 insulin like growth factor binding protein 3 216268_s_at 182 JAG1 8.50E-03 1.90 jagged 1 212236_x_at 3872 KRT17 2.53E-02 1.90 keratin 17, type I 201416_at 6659 SOX4 1.80E-03 1.90 SRY box 4 204597_x_at 6781 STC1 8.49E-02 1.90 stanniocalcin 1 231899_at 85463 ZC3H12C 2.80E-03 1.90 zinc finger CCCH-type containing 12C 204679_at 3775 KCNK1 1.20E-03 1.89 potassium channel, two pore domain subfamily K, member 1 235242_at 5966 REL 5.90E-03 1.89 v-rel avian reticuloendotheliosis viral oncogene homolog 230493_at 387914 SHISA2 2.70E-03 1.89 shisa family member 2 203888_at 7056 THBD 4.19E-02 1.89 thrombomodulin 227405_s_at 8325 FZD8 5.57E-02 1.88 frizzled class receptor 8 205992_s_at 3600 IL15 4.20E-03 1.88 interleukin 15 224571_at 359948 IRF2BP2 4.20E-03 1.88 interferon regulatory factor 2 binding protein 2 206706_at 4908 NTF3 3.80E-03 1.88 neurotrophin 3 231823_s_at 285590 SH3PXD2B 3.06E-02 1.88 SH3 and PX domains 2B 203373_at 8835 SOCS2 1.21E-02 1.88 suppressor of cytokine signaling 2 213003_s_at 23514 SPIDR 6.71E-02 1.88 scaffolding protein involved in DNA repair 226208_at 57688 ZSWIM6 5.20E-03 1.88 zinc finger, SWIM-type containing 6 203003_at 4209 MEF2D 1.05E-02 1.87 myocyte enhancer factor 2D 226140_s_at 220213 OTUD1 3.00E-04 1.87 OTU deubiquitinase 1 9586 CREB5 cAMP responsive element binding protein 5 242329_at 9.69E-02 1.86 401317 LOC401317 uncharacterized LOC401317 204015_s_at 1846 DUSP4 4.30E-03 1.86 dual specificity phosphatase 4 239058_at 2303 FOXC2 2.21E-02 1.86 forkhead box C2 201939_at 10769 PLK2 9.00E-04 1.86 polo-like kinase 2 1553965_x_at 388 RHOB 1.53E-02 1.86 ras homolog family member B 222783_s_at 64093 SMOC1 1.05E-02 1.86 SPARC related modular calcium binding 1 244007_at 58499 ZNF462 1.60E-03 1.86 zinc finger protein 462 211302_s_at 5142 PDE4B 1.39E-01 1.85 phosphodiesterase 4B, cAMP-specific 235849_at 286133 SCARA5 6.50E-03 1.85 scavenger receptor class A, member 5 202897_at 140885 SIRPA 2.18E-02 1.85 signal-regulatory protein alpha 229529_at 6943 TCF21 9.40E-03 1.85 transcription factor 21 216379_x_at 100133941 CD24 8.00E-04 1.84 CD24 molecule 204602_at 22943 DKK1 5.26E-02 1.84 dickkopf WNT signaling pathway inhibitor 1 227354_at 55824 PAG1 7.54E-02 1.84 phosphoprotein membrane anchor with glycosphingolipid microdomains 1 222258_s_at 23677 SH3BP4 1.10E-03 1.84 SH3-domain binding protein 4 202935_s_at 6662 SOX9 2.55E-02 1.84 SRY box 9 224963_at 1836 SLC26A2 1.60E-03 1.83 solute carrier family 26 (anion exchanger), member 2 205294_at 10458 BAIAP2 1.30E-03 1.82 BAI1-associated protein 2 209099_x_at 182 JAG1 4.50E-03 1.82 jagged 1 206765_at 3759 KCNJ2 3.48E-02 1.82 potassium channel, inwardly rectifying subfamily J, member 2 203180_at 220 ALDH1A3 6.85E-02 1.81 aldehyde dehydrogenase 1 family, member A3 208651_x_at 100133941 CD24 3.60E-02 1.81 CD24 molecule 232026_at 26091 HERC4 1.44E-01 1.81 HECT and RLD domain containing E3 ubiquitin protein ligase 4 229800_at 9201 DCLK1 1.00E-03 1.80 doublecortin-like kinase 1 228501_at 117248 GALNT15 1.84E-02 1.80 polypeptide N-acetylgalactosaminyltransferase 15 204698_at 3669 ISG20 2.75E-02 1.80 interferon stimulated exonuclease gene 20kDa 212831_at 1955 MEGF9 8.50E-03 1.80 multiple EGF-like-domains 9 217678_at 23657 SLC7A11 6.00E-04 1.80 solute carrier family 7 (anionic amino acid transporter light chain, xc- system), member 11 202936_s_at 6662 SOX9 7.10E-03 1.80 SRY box 9 204908_s_at 602 BCL3 2.90E-02 1.79 B-cell CLL/lymphoma 3 201926_s_at 1604 CD55 3.07E-02 1.79 CD55 molecule, decay accelerating factor for complement (Cromer blood group) 204678_s_at 3775 KCNK1 5.08E-02 1.79 potassium channel, two pore domain subfamily K, member 1 1557578_at 90102 PHLDB2 1.27E-01 1.79 pleckstrin homology-like domain, family B, member 2 1554822_at 57157 PHTF2 8.10E-03 1.79 putative homeodomain transcription factor 2 224973_at 55603 FAM46A 7.89E-02 1.78 family with sequence similarity 46, member A 204472_at 2669 GEM 6.90E-03 1.78 GTP binding protein overexpressed in skeletal muscle 203394_s_at 3280 HES1 5.78E-02 1.78 hes family bHLH transcription factor 1 226164_x_at 57494 RIMKLB 8.37E-02 1.78 ribosomal modification protein rimK-like family member B 266_s_at 100133941 CD24 4.13E-02 1.77 CD24 molecule 228759_at 64764 CREB3L2 1.74E-02 1.77 cAMP responsive element binding protein 3-like 2 221766_s_at 55603 FAM46A 4.30E-03 1.77 family with sequence similarity 46, member A 223380_s_at 26524 LATS2 1.10E-03 1.77 large tumor suppressor kinase 2 237724_at 162333 MARCH10 5.94E-02 1.76 membrane associated ring finger 10 203044_at 22856 CHSY1 5.00E-04 1.76 chondroitin sulfate synthase 1 227755_at 2081 ERN1 1.50E-03 1.76 endoplasmic reticulum to nucleus signaling 1 219889_at 10023 FRAT1 1.05E-02 1.76 frequently rearranged in advanced T-cell lymphomas 1 227032_at 5362 PLXNA2 5.04E-02 1.76 plexin A2 204197_s_at 864 RUNX3 9.90E-03 1.76 runt-related transcription factor 3 238029_s_at 151473 SLC16A14 3.00E-03 1.76 solute carrier family 16, member 14 202387_at 573 BAG1 7.00E-04 1.75 BCL2-associated athanogene 236361_at 117248 GALNT15 1.73E-02 1.75 polypeptide N-acetylgalactosaminyltransferase 15 218469_at 26585 GREM1 1.60E-03 1.75 gremlin 1, DAN family BMP antagonist 224569_s_at 359948 IRF2BP2 3.70E-03 1.75 interferon regulatory factor 2 binding protein 2 204684_at 4884 NPTX1 6.04E-02 1.75 neuronal pentraxin I 202861_at 5187 PER1 2.77E-02 1.75 period circadian clock 1 209771_x_at 100133941 CD24 6.00E-04 1.74 CD24 molecule 225978_at 57494 RIMKLB 1.50E-03 1.74 ribosomal modification protein rimK-like family member B 201418_s_at 6659 SOX4 1.02E-02 1.74 SRY box 4 230824_at 162333 MARCH10 1.79E-02 1.73 membrane associated ring finger 10 206374_at 1850 DUSP8 2.83E-02 1.73 dual specificity phosphatase 8 218696_at 9451 EIF2AK3 6.98E-02 1.73 eukaryotic translation initiation factor 2-alpha kinase 3 201329_s_at 2114 ETS2 1.50E-03 1.73 v-ets avian erythroblastosis virus E26 oncogene homolog 2 218468_s_at 26585 GREM1 9.00E-04 1.73 gremlin 1, DAN family BMP antagonist 226333_at 3570 IL6R 4.04E-02 1.73 interleukin 6 receptor 209098_s_at 182 JAG1 1.89E-02 1.73 jagged 1 220266_s_at 9314 KLF4 7.49E-02 1.73 Kruppel-like factor 4 (gut) 206750_at 7975 MAFK 2.02E-02 1.73 v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog K 200730_s_at 7803 PTP4A1 8.50E-03 1.73 protein tyrosine phosphatase type IVA, member 1 203439_s_at 8614 STC2 1.00E-03 1.73 stanniocalcin 2 218182_s_at 9076 CLDN1 1.06E-01 1.72 claudin 1 205035_at 9150 CTDP1 1.08E-02 1.72 CTD phosphatase subunit 1 224657_at 54206 ERRFI1 4.10E-03 1.72 ERBB receptor feedback inhibitor 1 204011_at 10253 SPRY2 1.69E-02 1.72 sprouty RTK signaling antagonist 2 220400_at 157680 VPS13B 1.17E-01 1.72 vacuolar protein sorting 13 homolog B (yeast) 222549_at 9076 CLDN1 4.70E-03 1.71 claudin 1 210350_x_at 3621 ING1 4.14E-02 1.71 inhibitor of growth family member 1 231559_at 4837 NNMT 1.79E-02 1.71 nicotinamide N-methyltransferase 202679_at 4864 NPC1 3.00E-03 1.71 Niemann-Pick disease, type C1 222662_at 79660 PPP1R3B 3.79E-02 1.71 protein phosphatase 1, regulatory subunit 3B 239615_at 6584 SLC22A5 6.40E-03 1.71 solute carrier family 22 (organic cation/carnitine transporter), member 5 209921_at 23657 SLC7A11 6.00E-04 1.71 solute carrier family 7 (anionic amino acid transporter light chain, xc- system), member 11 238542_at 80328 ULBP2 1.60E-03 1.71 UL16 binding protein 2 202628_s_at 5054 SERPINE1 1.50E-03 1.70 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 213624_at 10924 SMPDL3A 4.45E-02 1.70 sphingomyelin phosphodiesterase, acid-like 3A 213668_s_at 6659 SOX4 3.35E-02 1.70 SRY box 4 218810_at 80149 ZC3H12A 3.60E-02 1.70 zinc finger CCCH-type containing 12A 203973_s_at 1052 CEBPD 4.00E-04 1.69 CCAAT/enhancer binding protein (C/EBP), delta 242767_at 29995 LMCD1 1.65E-02 1.69 LIM and cysteine-rich domains 1 202431_s_at 4609 MYC 3.90E-03 1.69 v-myc avian myelocytomatosis viral oncogene homolog 230330_at 8493 PPM1D 2.06E-02 1.69 protein phosphatase, Mg2+/Mn2+ dependent, 1D 204595_s_at 6781 STC1 3.52E-02 1.69 stanniocalcin 1 221577_x_at 9518 GDF15 1.00E-02 1.68 growth differentiation factor 15 201195_s_at 8140 SLC7A5 2.11E-02 1.68 solute carrier family 7 (amino acid transporter light chain, L system), member 5 202181_at 9766 SUSD6 2.70E-03 1.68 sushi domain containing 6 227195_at 84858 ZNF503 1.87E-02 1.68 zinc finger protein 503 204039_at 1050 CEBPA 7.58E-02 1.67 CCAAT/enhancer binding protein (C/EBP), alpha 241985_at 133746 JMY 1.35E-02 1.67 junction mediating and regulatory protein, p53 cofactor 201810_s_at 9467 SH3BP5 3.00E-03 1.67 SH3-domain binding protein 5 (BTK-associated) 211475_s_at 573 BAG1 9.00E-04 1.66 BCL2-associated athanogene 1552487_a_at 646 BNC1 5.20E-03 1.66 basonuclin 1 212830_at 1955 MEGF9 4.30E-03 1.66 multiple EGF-like-domains 9 203438_at 8614 STC2 2.02E-02 1.66 stanniocalcin 2 204931_at 6943 TCF21 3.10E-03 1.66 transcription factor 21 209386_at 4071 TM4SF1 7.20E-03 1.66 transmembrane 4 L six family member 1 157693 FAM87A family with sequence similarity 87, member A 1559140_at 7.49E-02 1.65 400728 FAM87B family with sequence similarity 87, member B 206997_s_at 9394 HS6ST1 1.30E-01 1.65 heparan sulfate 6-O-sulfotransferase 1 204198_s_at 864 RUNX3 2.13E-02 1.65 runt-related transcription factor 3 206511_s_at 10736 SIX2 1.55E-02 1.65 SIX homeobox 2 224126_at 84068 SLC10A7 9.42E-02 1.65 solute carrier family 10, member 7 225615_at 126917 IFFO2 1.04E-01 1.64 intermediate filament family orphan 2 204967_at 357 SHROOM2 1.18E-01 1.64 shroom family member 2 204653_at 7020 TFAP2A 3.10E-03 1.64 transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha) 235404_at 84159 ARID5B 1.09E-01 1.63 AT rich interactive domain 5B (MRF1-like) 214445_at 22936 ELL2 1.34E-01 1.63 elongation factor, RNA polymerase II, 2 209864_at 23401 FRAT2 1.43E-02 1.63 frequently rearranged in advanced T-cell lymphomas 2 204512_at 3096 HIVEP1 9.40E-03 1.63 human immunodeficiency virus type I enhancer binding protein 1 212641_at 3097 HIVEP2 2.27E-02 1.63 human immunodeficiency virus type I enhancer binding protein 2 225263_at 9394 HS6ST1 9.40E-03 1.63 heparan sulfate 6-O-sulfotransferase 1 202464_s_at 5209 PFKFB3 6.30E-03 1.63 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 225056_at 57568 SIPA1L2 6.39E-02 1.63 signal-induced proliferation-associated 1 like 2 212614_at 84159 ARID5B 2.20E-03 1.62 AT rich interactive domain 5B (MRF1-like) 219825_at 56603 CYP26B1 1.48E-01 1.62 cytochrome P450, family 26, subfamily B, polypeptide 1 235122_at 59269 HIVEP3 3.98E-02 1.62 human immunodeficiency virus type I enhancer binding protein 3 217371_s_at 3600 IL15 8.39E-02 1.62 interleukin 15 205945_at 3570 IL6R 1.99E-02 1.62 interleukin 6 receptor 209808_x_at 3621 ING1 5.60E-03 1.62 inhibitor of growth family member 1 33304_at 3669 ISG20 2.93E-02 1.62 interferon stimulated exonuclease gene 20kDa 225688_s_at 90102 PHLDB2 9.83E-02 1.62 pleckstrin homology-like domain, family B, member 2 204286_s_at 5366 PMAIP1 9.50E-02 1.62 phorbol-12-myristate-13-acetate-induced protein 1 205097_at 1836 SLC26A2 5.20E-03 1.62 solute carrier family 26 (anion exchanger), member 2 227867_at 129293 TRABD2A 3.98E-02 1.62 TraB domain containing 2A 210511_s_at 3624 INHBA 5.57E-02 1.61 inhibin beta A 211205_x_at 8394 PIP5K1A 1.60E-02 1.61 phosphatidylinositol-4-phosphate 5-kinase, type I, alpha 229344_x_at 57494 RIMKLB 3.63E-02 1.61 ribosomal modification protein rimK-like family member B 201417_at 6659 SOX4 3.80E-03 1.61 SRY box 4 203220_s_at 7088 TLE1 5.26E-02 1.61 transducin-like enhancer of split 1 (E(sp1) homolog, Drosophila) 243259_at 6314 ATXN7 5.79E-02 1.60 ataxin 7 233506_at 9689 BZW1 4.39E-02 1.60 basic leucine zipper and W2 domains 1 214595_at 3755 KCNG1 5.38E-02 1.60 potassium channel, voltage gated modifier subfamily G, member 1 202883_s_at 5519 PPP2R1B 2.33E-02 1.60 protein phosphatase 2, regulatory subunit A, beta 226101_at 5581 PRKCE 7.10E-03 1.60 protein kinase C, epsilon 213988_s_at 6303 SAT1 7.21E-02 1.60 spermidine/spermine N1-acetyltransferase 1 226492_at 80031 SEMA6D 3.05E-02 1.60 sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6D 207332_s_at 7037 TFRC 1.09E-02 1.60 transferrin receptor 209387_s_at 4071 TM4SF1 4.82E-02 1.60 transmembrane 4 L six family member 1 1555007_s_at 144406 WDR66 2.10E-03 1.60 WD repeat domain 66 1553096_s_at 10018 BCL2L11 2.70E-02 1.59 BCL2-like 11 (apoptosis facilitator) 206581_at 646 BNC1 5.90E-03 1.59 basonuclin 1 203628_at 3480 IGF1R 2.33E-02 1.59 insulin-like growth factor 1 receptor 1554833_at 55784 MCTP2 1.37E-02 1.59 multiple C2 domains, transmembrane 2 213963_s_at 8819 SAP30 1.30E-01 1.59 Sin3A associated protein 30kDa 208234_x_at 2263 FGFR2 1.16E-01 1.58 fibroblast growth factor receptor 2 202838_at 2517 FUCA1 6.90E-03 1.58 fucosidase, alpha-L- 1, tissue 205227_at 3556 IL1RAP 3.40E-02 1.58 interleukin 1 receptor accessory protein 219657_s_at 51274 KLF3 5.04E-02 1.58 Kruppel-like factor 3 (basic) 229092_at 7026 NR2F2 9.40E-03 1.58 nuclear receptor subfamily 2, group F, member 2 202886_s_at 5519 PPP2R1B 3.98E-02 1.58 protein phosphatase 2, regulatory subunit A, beta 237215_s_at 7037 TFRC 1.11E-01 1.58 transferrin receptor 228404_at 153572 IRX2 1.39E-01 1.57 iroquois homeobox 2 212272_at 23175 LPIN1 6.14E-02 1.57 lipin 1 209758_s_at 8076 MFAP5 3.33E-02 1.57 microfibrillar associated protein 5 202238_s_at 4837 NNMT 1.05E-02 1.57 nicotinamide N-methyltransferase 36829_at 5187 PER1 6.70E-03 1.57 period circadian clock 1 216074_x_at 23286 WWC1 1.21E-02 1.57 WW and C2 domain containing 1 241969_at 84159 ARID5B 8.15E-02 1.56 AT rich interactive domain 5B (MRF1-like) 200920_s_at 694 BTG1 5.60E-03 1.56 B-cell translocation gene 1, anti-proliferative 1553134_s_at 203228 C9orf72 8.79E-02 1.56 chromosome 9 open reading frame 72 224822_at 10395 DLC1 4.00E-03 1.56 DLC1 Rho GTPase activating protein 222866_s_at 55640 FLVCR2 1.17E-02 1.56 feline leukemia virus subgroup C cellular receptor family, member 2 230492_s_at 56261 GPCPD1 6.02E-02 1.56 glycerophosphocholine phosphodiesterase 1 212642_s_at 3097 HIVEP2 1.19E-02 1.56 human immunodeficiency virus type I enhancer binding protein 2 204952_at 27076 LYPD3 1.04E-01 1.56 LY6/PLAUR domain containing 3 223218_s_at 64332 NFKBIZ 1.27E-01 1.56 nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta 201811_x_at 9467 SH3BP5 2.90E-03 1.56 SH3-domain binding protein 5 (BTK-associated) 228181_at 7779 SLC30A1 1.08E-01 1.56 solute carrier family 30 (zinc transporter), member 1 203221_at 7088 TLE1 3.05E-02 1.56 transducin-like enhancer of split 1 (E(sp1) homolog, Drosophila) 224978_s_at 57602 USP36 9.13E-02 1.56 ubiquitin specific peptidase 36 226982_at 22936 ELL2 2.12E-02 1.55 elongation factor, RNA polymerase II, 2 200879_s_at 2034 EPAS1 1.09E-01 1.55 endothelial PAS domain protein 1 214212_x_at 10979 FERMT2 1.64E-02 1.55 fermitin family member 2 243358_at 3480 IGF1R 4.20E-03 1.55 insulin-like growth factor 1 receptor 217489_s_at 3570 IL6R 1.34E-01 1.55 interleukin 6 receptor 203542_s_at 687 KLF9 2.90E-03 1.55 Kruppel-like factor 9 226844_at 79817 MOB3B 6.55E-02 1.55 MOB kinase activator 3B 238919_at 5101 PCDH9 1.46E-01 1.55 protocadherin 9 204285_s_at 5366 PMAIP1 2.30E-02 1.55 phorbol-12-myristate-13-acetate-induced protein 1 213280_at 23108 RAP1GAP2 6.00E-03 1.55 RAP1 GTPase activating protein 2 220924_s_at 54407 SLC38A2 1.30E-03 1.55 solute carrier family 38, member 2 225033_at 6482 ST3GAL1 4.92E-02 1.55 ST3 beta-galactoside alpha-2,3-sialyltransferase 1 204596_s_at 6781 STC1 8.05E-02 1.55 stanniocalcin 1 202644_s_at 7128 TNFAIP3 7.49E-02 1.55 tumor necrosis factor, alpha-induced protein 3 209946_at 7424 VEGFC 1.51E-02 1.55 vascular endothelial growth factor C 209964_s_at 6314 ATXN7 1.17E-01 1.54 ataxin 7 214014_at 10435 CDC42EP2 2.21E-02 1.54 CDC42 effector protein (Rho GTPase binding) 2 208072_s_at 8527 DGKD 1.62E-02 1.54 diacylglycerol kinase, delta 130kDa 1554966_a_at 11259 FILIP1L 2.75E-02 1.54 filamin A interacting protein 1-like 230788_at 2651 GCNT2 1.74E-02 1.54 glucosaminyl (N-acetyl) transferase 2, I-branching enzyme (I blood group) 203821_at 1839 HBEGF 7.30E-03 1.54 heparin-binding EGF-like growth factor 208960_s_at 1316 KLF6 2.30E-03 1.54 Kruppel-like factor 6 861 RUNX1 runt-related transcription factor 1 210805_x_at 8.70E-03 1.54 100506403 LOC100506403 uncharacterized LOC100506403 202896_s_at 140885 SIRPA 1.09E-02 1.54 signal-regulatory protein alpha 212290_at 6541 SLC7A1 6.23E-02 1.54 solute carrier family 7 (cationic amino acid transporter, y+ system), member 1 221291_at 80328 ULBP2 3.12E-02 1.54 UL16 binding protein 2 236557_at 253461 ZBTB38 2.75E-02 1.54 zinc finger and BTB domain containing 38 218865_at 64757 MARC1 5.90E-03 1.53 mitochondrial amidoxime reducing component 1 212974_at 22898 DENND3 1.23E-01 1.53 DENN/MADD domain containing 3 203592_s_at 10272 FSTL3 7.79E-02 1.53 follistatin-like 3 (secreted glycoprotein) 203097_s_at 9693 RAPGEF2 2.30E-03 1.53 Rap guanine nucleotide exchange factor 2 203096_s_at 9693 RAPGEF2 1.59E-02 1.53 Rap guanine nucleotide exchange factor 2 209481_at 54861 SNRK 3.00E-03 1.53 SNF related kinase 203759_at 6484 ST3GAL4 2.19E-02 1.53 ST3 beta-galactoside alpha-2,3-sialyltransferase 4 205990_s_at 7474 WNT5A 2.83E-02 1.53 wingless-type MMTV integration site family, member 5A 210517_s_at 9590 AKAP12 1.14E-02 1.52 A kinase (PRKA) anchor protein 12 201508_at 3487 IGFBP4 3.82E-02 1.52 insulin like growth factor binding protein 4 208415_x_at 3621 ING1 8.80E-03 1.52 inhibitor of growth family member 1 231849_at 144501 KRT80 8.60E-03 1.52 keratin 80, type II 212823_s_at 26030 PLEKHG3 6.70E-02 1.52 pleckstrin homology domain containing, family G (with RhoGef domain) member 3 227093_at 57602 USP36 1.17E-01 1.52 ubiquitin specific peptidase 36 241950_at 23286 WWC1 1.27E-01 1.52 WW and C2 domain containing 1 210762_s_at 10395 DLC1 8.60E-03 1.51 DLC1 Rho GTPase activating protein 203627_at 3480 IGF1R 6.88E-02 1.51 insulin-like growth factor 1 receptor 233487_s_at 56262 LRRC8A 6.00E-03 1.51 leucine rich repeat containing 8 family, member A 210845_s_at 5329 PLAUR 4.00E-03 1.51 plasminogen activator, urokinase receptor 216049_at 22836 RHOBTB3 6.50E-02 1.51 Rho-related BTB domain containing 3 226563_at 4087 SMAD2 3.93E-02 1.51 SMAD family member 2 235598_at 4087 SMAD2 6.82E-02 1.51 SMAD family member 2 213873_at 10402 ST3GAL6 7.38E-02 1.51 ST3 beta-galactoside alpha-2,3-sialyltransferase 6 229812_at 84196 USP48 4.92E-02 1.51 ubiquitin specific peptidase 48

Indacaterol (6h) (320 Probe Sets ≡ 205 Gene IDs) Affy Probe Set ID* Entrez Gene ID Official Gene Symbol FDR P-Value Fold change UniGene Name Function 209959_at 8013 NR4A3 5.00E-04 14.13 nuclear receptor subfamily 4, group A, member 3 221541_at 83716 CRISPLD2 5.31E-08 8.05 cysteine-rich secretory protein LCCL domain containing 2 1565657_at 54918 CMTM6 9.35E-02 6.78 CKLF-like MARVEL transmembrane domain containing 6 219049_at 55790 CSGALNACT1 3.00E-04 6.58 chondroitin sulfate N-acetylgalactosaminyltransferase 1 228128_x_at 5069 PAPPA 1.00E-04 6.16 pregnancy-associated plasma protein A, pappalysin 1 222784_at 64093 SMOC1 1.00E-04 5.62 SPARC related modular calcium binding 1 225207_at 5166 PDK4 7.00E-04 5.41 pyruvate dehydrogenase kinase, isozyme 4 239461_at 117248 GALNT15 4.05E-02 5.02 polypeptide N-acetylgalactosaminyltransferase 15 222783_s_at 64093 SMOC1 1.00E-04 4.93 SPARC related modular calcium binding 1 243438_at 27115 PDE7B 8.50E-03 4.85 phosphodiesterase 7B 205421_at 6581 SLC22A3 5.51E-02 4.77 solute carrier family 22 (organic cation transporter), member 3 1556185_a_at 79689 STEAP4 5.00E-04 4.73 STEAP family member 4 224942_at 5069 PAPPA 3.00E-04 4.69 pregnancy-associated plasma protein A, pappalysin 1 224940_s_at 5069 PAPPA 3.00E-04 4.68 pregnancy-associated plasma protein A, pappalysin 1 203638_s_at 2263 FGFR2 1.40E-03 4.65 fibroblast growth factor receptor 2 224941_at 5069 PAPPA 5.00E-04 4.64 pregnancy-associated plasma protein A, pappalysin 1 228962_at 5144 PDE4D 1.30E-03 4.51 phosphodiesterase 4D, cAMP-specific 230109_at 27115 PDE7B 3.00E-04 4.31 phosphodiesterase 7B 201981_at 5069 PAPPA 7.00E-04 4.10 pregnancy-associated plasma protein A, pappalysin 1 238649_at 26207 PITPNC1 2.20E-03 4.10 phosphatidylinositol transfer protein, cytoplasmic 1 228501_at 117248 GALNT15 1.60E-03 4.02 polypeptide N-acetylgalactosaminyltransferase 15 203549_s_at 4023 LPL 3.00E-04 3.99 lipoprotein lipase 211401_s_at 2263 FGFR2 4.96E-02 3.85 fibroblast growth factor receptor 2 203548_s_at 4023 LPL 1.00E-03 3.82 lipoprotein lipase 213050_at 23242 COBL 5.00E-04 3.79 cordon-bleu WH2 repeat protein 220088_at 728 C5AR1 9.00E-04 3.65 complement component 5a receptor 1 225987_at 79689 STEAP4 3.00E-04 3.44 STEAP family member 4 211119_at 2100 ESR2 3.77E-02 3.44 estrogen receptor 2 (ER beta) 236361_at 117248 GALNT15 6.20E-03 3.41 polypeptide N-acetylgalactosaminyltransferase 15 237804_at 8701 DNAH11 4.79E-02 3.41 dynein, axonemal, heavy chain 11 205960_at 5166 PDK4 2.24E-02 3.40 pyruvate dehydrogenase kinase, isozyme 4 229709_at 483 ATP1B3 8.00E-03 3.39 ATPase, Na+/K+ transporting, beta 3 polypeptide 216248_s_at 4929 NR4A2 6.53E-02 3.39 nuclear receptor subfamily 4, group A, member 2 225516_at 6542 SLC7A2 1.00E-04 3.33 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 208606_s_at 54361 WNT4 6.96E-02 3.27 wingless-type MMTV integration site family, member 4 213182_x_at 1028 CDKN1C 3.10E-03 3.14 cyclin-dependent kinase inhibitor 1C (p57, Kip2) 205501_at 10846 PDE10A 5.43E-05 3.00 phosphodiesterase 10A 220540_at 60598 KCNK15 2.79E-02 2.93 potassium channel, two pore domain subfamily K, member 15 219093_at 55022 PID1 3.90E-03 2.86 phosphotyrosine interaction domain containing 1 204491_at 5144 PDE4D 1.03E-02 2.82 phosphodiesterase 4D, cAMP-specific 219534_x_at 1028 CDKN1C 1.40E-03 2.75 cyclin-dependent kinase inhibitor 1C (p57, Kip2) 216894_x_at 1028 CDKN1C 3.00E-04 2.74 cyclin-dependent kinase inhibitor 1C (p57, Kip2) 213348_at 1028 CDKN1C 9.00E-04 2.73 cyclin-dependent kinase inhibitor 1C (p57, Kip2) 220187_at 79689 STEAP4 7.00E-04 2.70 STEAP family member 4 223484_at 84419 C15orf48 9.39E-02 2.69 chromosome 15 open reading frame 48 239650_at 344148 NCKAP5 1.11E-02 2.68 NCK-associated protein 5 237939_at 2044 EPHA5 3.00E-04 2.64 EPH receptor A5 230748_at 9120 SLC16A6 1.63E-02 2.55 solute carrier family 16, member 6 207038_at 9120 SLC16A6 1.72E-02 2.49 solute carrier family 16, member 6 1454 CSNK1E casein kinase 1, epsilon 222015_at 5.00E-04 2.45 102800317 LOC400927-CSNK1E LOC400927-CSNK1E readthrough 216627_s_at 2683 B4GALT1 2.40E-03 2.44 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1 210102_at 4013 VWA5A 2.93E-02 2.41 von Willebrand factor A domain containing 5A 229093_at 4846 NOS3 6.78E-02 2.38 nitric oxide synthase 3 228132_at 84448 ABLIM2 3.00E-04 2.36 actin binding LIM protein family, member 2 236300_at 5139 PDE3A 1.30E-03 2.35 phosphodiesterase 3A, cGMP-inhibited 238029_s_at 151473 SLC16A14 1.90E-03 2.35 solute carrier family 16, member 14 210836_x_at 5144 PDE4D 1.06E-02 2.35 phosphodiesterase 4D, cAMP-specific 1555007_s_at 144406 WDR66 3.00E-04 2.34 WD repeat domain 66 228507_at 5139 PDE3A 2.20E-03 2.34 phosphodiesterase 3A, cGMP-inhibited 208228_s_at 2263 FGFR2 2.60E-03 2.32 fibroblast growth factor receptor 2 209583_s_at 4345 CD200 2.80E-03 2.32 CD200 molecule 229414_at 26207 PITPNC1 3.25E-02 2.32 phosphatidylinositol transfer protein, cytoplasmic 1 1553199_at 138009 DCAF4L2 6.02E-02 2.31 DDB1 and CUL4 associated factor 4-like 2 224071_at 50604 IL20 6.00E-02 2.30 interleukin 20 225978_at 57494 RIMKLB 3.00E-04 2.29 ribosomal modification protein rimK-like family member B 1555006_at 144406 WDR66 1.11E-02 2.28 WD repeat domain 66 54576 UGT1A8 UDP glucuronosyltransferase 1 family, polypeptide A8 221305_s_at 9.20E-02 2.27 54600 UGT1A9 UDP glucuronosyltransferase 1 family, polypeptide A9 8739 HRK harakiri, BCL2 interacting protein 229552_at 1.20E-03 2.24 283454 LOC283454 uncharacterized LOC283454 208425_s_at 26115 TANC2 1.20E-03 2.24 tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 209582_s_at 4345 CD200 1.20E-03 2.22 CD200 molecule 232267_at 283383 ADGRD1 8.60E-03 2.22 adhesion G protein-coupled receptor D1 206616_s_at 53616 ADAM22 2.07E-02 2.22 ADAM metallopeptidase domain 22 1552736_a_at 81832 NETO1 3.00E-04 2.21 neuropilin (NRP) and tolloid (TLL)-like 1 1554199_at 5800 PTPRO 4.44E-02 2.20 protein tyrosine phosphatase, receptor type, O 218177_at 57132 CHMP1B 2.94E-02 2.18 charged multivesicular body protein 1B 205003_at 9732 DOCK4 1.92E-02 2.17 dedicator of cytokinesis 4 227099_s_at 387763 C11orf96 1.80E-03 2.16 chromosome 11 open reading frame 96 206389_s_at 5139 PDE3A 2.40E-03 2.16 phosphodiesterase 3A, cGMP-inhibited 209772_s_at 100133941 CD24 1.00E-04 2.15 CD24 molecule 206356_s_at 2774 GNAL 1.20E-03 2.15 guanine nucleotide binding protein, alpha activating activity polypeptide, olfactory type 218589_at 10161 LPAR6 1.13E-02 2.15 lysophosphatidic acid receptor 6 228325_at 23514 SPIDR 4.55E-02 2.15 scaffolding protein involved in DNA repair 235367_at 84665 MYPN 9.00E-04 2.13 myopalladin 213006_at 1052 CEBPD 7.22E-02 2.13 CCAAT/enhancer binding protein (C/EBP), delta 224952_at 26115 TANC2 3.00E-04 2.12 tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 203628_at 3480 IGF1R 9.00E-04 2.10 insulin-like growth factor 1 receptor 213436_at 1268 CNR1 1.30E-03 2.10 cannabinoid receptor 1 (brain) 218865_at 64757 MARC1 3.00E-04 2.09 mitochondrial amidoxime reducing component 1 207626_s_at 6542 SLC7A2 1.55E-02 2.09 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 220335_x_at 23491 CES3 3.00E-04 2.08 carboxylesterase 3 205157_s_at 3872 KRT17 1.63E-02 2.08 keratin 17, type I 230398_at 84951 TNS4 4.36E-02 2.07 tensin 4 205896_at 6583 SLC22A4 9.20E-03 2.06 solute carrier family 22 (organic cation/zwitterion transporter), member 4 150094 SIK1 salt-inducible kinase 1 208078_s_at 7.78E-02 2.06 102724428 LOC102724428 serine/threonine-protein kinase SIK1 208651_x_at 100133941 CD24 2.05E-02 2.05 CD24 molecule 1568949_at 26207 PITPNC1 8.20E-03 2.03 phosphatidylinositol transfer protein, cytoplasmic 1 206359_at 9021 SOCS3 4.68E-02 2.03 suppressor of cytokine signaling 3 207430_s_at 4477 MSMB 9.32E-02 2.03 microseminoprotein, beta- 208650_s_at 100133941 CD24 8.00E-03 2.02 CD24 molecule 201810_s_at 9467 SH3BP5 1.20E-03 2.01 SH3-domain binding protein 5 (BTK-associated) 1454 CSNK1E casein kinase 1, epsilon 226858_at 5.00E-04 2.00 102800317 LOC400927-CSNK1E LOC400927-CSNK1E readthrough 224963_at 1836 SLC26A2 9.00E-04 2.00 solute carrier family 26 (anion exchanger), member 2 236440_at 81832 NETO1 1.30E-03 2.00 neuropilin (NRP) and tolloid (TLL)-like 1 211631_x_at 2683 B4GALT1 3.50E-03 1.99 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1 206864_s_at 8739 HRK 3.00E-04 1.98 harakiri, BCL2 interacting protein 236087_at 84448 ABLIM2 1.20E-03 1.98 actin binding LIM protein family, member 2 266_s_at 100133941 CD24 2.84E-02 1.98 CD24 molecule 210837_s_at 5144 PDE4D 7.29E-02 1.98 phosphodiesterase 4D, cAMP-specific 237724_at 162333 MARCH10 2.61E-02 1.97 membrane associated ring finger 10 206388_at 5139 PDE3A 5.00E-04 1.96 phosphodiesterase 3A, cGMP-inhibited 226865_at 84898 PLXDC2 1.40E-03 1.96 plexin domain containing 2 242444_at 114904 C1QTNF6 1.31E-02 1.96 C1q and tumor necrosis factor related protein 6 238919_at 5101 PCDH9 3.88E-02 1.95 protocadherin 9 200731_s_at 7803 PTP4A1 5.85E-02 1.95 protein tyrosine phosphatase type IVA, member 1 224393_s_at 27439 TMEM121B 8.80E-02 1.95 transmembrane Protein 121B 225728_at 8470 SORBS2 1.57E-02 1.94 sorbin and SH3 domain containing 2 203627_at 3480 IGF1R 9.00E-04 1.93 insulin-like growth factor 1 receptor 213438_at 23114 NFASC 1.30E-03 1.93 neurofascin 215703_at 1080 CFTR 8.96E-02 1.92 cystic fibrosis transmembrane conductance regulator 213765_at 8076 MFAP5 1.20E-03 1.91 microfibrillar associated protein 5 202952_s_at 8038 ADAM12 3.00E-04 1.90 ADAM metallopeptidase domain 12 220343_at 27115 PDE7B 3.00E-04 1.89 phosphodiesterase 7B 214807_at 84898 PLXDC2 1.63E-02 1.89 plexin domain containing 2 219155_at 26207 PITPNC1 1.93E-02 1.89 phosphatidylinositol transfer protein, cytoplasmic 1 206865_at 8739 HRK 3.31E-02 1.89 harakiri, BCL2 interacting protein 1559997_x_at 201191 SAMD14 6.77E-02 1.89 sterile alpha motif domain containing 14 216379_x_at 100133941 CD24 3.10E-03 1.88 CD24 molecule 210397_at 1672 DEFB1 7.30E-03 1.88 defensin, beta 1 228964_at 639 PRDM1 1.71E-02 1.88 PR domain containing 1, with ZNF domain 225330_at 3480 IGF1R 4.50E-02 1.88 insulin-like growth factor 1 receptor 214595_at 3755 KCNG1 7.60E-03 1.87 potassium channel, voltage gated modifier subfamily G, member 1 223571_at 114904 C1QTNF6 1.35E-02 1.87 C1q and tumor necrosis factor related protein 6 226614_s_at 83648 FAM167A 3.00E-04 1.86 family with sequence similarity 167, member A 213924_at 2774 GNAL 8.20E-03 1.86 guanine nucleotide binding protein, alpha activating activity polypeptide, olfactory type 213764_s_at 8076 MFAP5 1.82E-02 1.86 microfibrillar associated protein 5 221272_s_at 81563 C1orf21 3.20E-03 1.85 chromosome 1 open reading frame 21 242162_at 164781 DAW1 8.60E-03 1.85 dynein assembly factor with WDR repeat domains 1 237187_at 8739 HRK 9.00E-04 1.84 harakiri, BCL2 interacting protein 227529_s_at 9590 AKAP12 1.72E-02 1.84 A kinase (PRKA) anchor protein 12 227276_at 84898 PLXDC2 1.30E-03 1.83 plexin domain containing 2 206355_at 2774 GNAL 1.60E-03 1.83 guanine nucleotide binding protein, alpha activating activity polypeptide, olfactory type 208250_s_at 1755 DMBT1 3.10E-03 1.83 deleted in malignant brain tumors 1 212236_x_at 3872 KRT17 4.52E-02 1.83 keratin 17, type I 1563933_a_at 200150 PLD5 5.00E-04 1.82 phospholipase D family, member 5 229180_at 23286 WWC1 1.30E-03 1.82 WW and C2 domain containing 1 209771_x_at 100133941 CD24 2.20E-03 1.82 CD24 molecule 237217_at 92949 ADAMTSL1 8.20E-03 1.82 ADAMTS like 1 219743_at 23493 HEY2 5.53E-02 1.82 hes-related family bHLH transcription factor with YRPW motif 2 202388_at 5997 RGS2 7.25E-02 1.82 regulator of G-protein signaling 2 201811_x_at 9467 SH3BP5 2.10E-03 1.81 SH3-domain binding protein 5 (BTK-associated) 225999_at 57494 RIMKLB 2.48E-02 1.81 ribosomal modification protein rimK-like family member B 218178_s_at 57132 CHMP1B 3.03E-02 1.81 charged multivesicular body protein 1B 209016_s_at 3855 KRT7 9.00E-04 1.80 keratin 7, type II 1562713_a_at 81832 NETO1 3.30E-03 1.80 neuropilin (NRP) and tolloid (TLL)-like 1 208335_s_at 2532 ACKR1 1.03E-02 1.80 atypical chemokine receptor 1 (Duffy blood group) 229403_at 2683 B4GALT1 7.27E-02 1.80 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 1 243243_at 2719 GPC3 1.48E-02 1.79 glypican 3 221577_x_at 9518 GDF15 2.43E-02 1.79 growth differentiation factor 15 1554744_at 114769 CARD16 2.64E-02 1.79 caspase recruitment domain family, member 16 200732_s_at 7803 PTP4A1 2.92E-02 1.79 protein tyrosine phosphatase type IVA, member 1 213425_at 7474 WNT5A 7.78E-02 1.79 wingless-type MMTV integration site family, member 5A 205139_s_at 10090 UST 8.29E-02 1.79 uronyl-2-sulfotransferase 1560253_at 56956 LHX9 8.92E-02 1.79 LIM homeobox 9 229529_at 6943 TCF21 9.00E-04 1.78 transcription factor 21 226777_at 8038 ADAM12 1.30E-03 1.78 ADAM metallopeptidase domain 12 225328_at 114907 FBXO32 1.70E-03 1.78 F-box protein 32 226834_at 79827 CLMP 6.30E-03 1.78 CXADR-like membrane protein 223475_at 83690 CRISPLD1 1.20E-03 1.77 cysteine-rich secretory protein LCCL domain containing 1 205097_at 1836 SLC26A2 1.50E-03 1.77 solute carrier family 26 (anion exchanger), member 2 215189_at 3892 KRT86 2.00E-03 1.77 keratin 86, type II 227752_at 55304 SPTLC3 3.10E-03 1.77 serine palmitoyltransferase, long chain base subunit 3 228748_at 966 CD59 1.56E-02 1.77 CD59 molecule, complement regulatory protein 236297_at 84898 PLXDC2 2.91E-02 1.77 plexin domain containing 2 227660_at 84168 ANTXR1 3.00E-04 1.76 anthrax toxin receptor 1 209921_at 23657 SLC7A11 2.07E-02 1.76 solute carrier family 7 (anionic amino acid transporter light chain, xc- system), member 11 209387_s_at 4071 TM4SF1 2.22E-02 1.76 transmembrane 4 L six family member 1 236038_at 57484 RNF150 3.89E-02 1.76 ring finger protein 150 214842_s_at 213 ALB 4.74E-02 1.76 albumin 220092_s_at 84168 ANTXR1 7.00E-04 1.75 anthrax toxin receptor 1 229720_at 573 BAG1 1.10E-03 1.75 BCL2-associated athanogene 203710_at 3708 ITPR1 6.40E-03 1.75 inositol 1,4,5-trisphosphate receptor, type 1 215303_at 9201 DCLK1 2.60E-03 1.74 doublecortin-like kinase 1 219383_at 79899 PRR5L 3.10E-03 1.74 proline rich 5 like 228731_at 2977 GUCY1A2 8.87E-02 1.74 guanylate cyclase 1, soluble, alpha 2 212143_s_at 3486 IGFBP3 3.80E-03 1.73 insulin like growth factor binding protein 3 230962_at 9201 DCLK1 7.30E-03 1.72 doublecortin-like kinase 1 225520_at 25902 MTHFD1L 8.00E-03 1.72 methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like 238455_at 84898 PLXDC2 1.15E-02 1.72 plexin domain containing 2 227530_at 9590 AKAP12 2.20E-02 1.72 A kinase (PRKA) anchor protein 12 236201_at 55304 SPTLC3 6.71E-02 1.72 serine palmitoyltransferase, long chain base subunit 3 241950_at 23286 WWC1 8.80E-03 1.71 WW and C2 domain containing 1 217678_at 23657 SLC7A11 1.40E-02 1.71 solute carrier family 7 (anionic amino acid transporter light chain, xc- system), member 11 202478_at 28951 TRIB2 2.78E-02 1.71 tribbles pseudokinase 2 212906_at 57476 GRAMD1B 9.87E-02 1.71 GRAM domain containing 1B 202897_at 140885 SIRPA 6.80E-03 1.70 signal-regulatory protein alpha 209277_at 7980 TFPI2 2.12E-02 1.70 tissue factor pathway inhibitor 2 231559_at 4837 NNMT 2.35E-02 1.70 nicotinamide N-methyltransferase 203180_at 220 ALDH1A3 1.20E-03 1.69 aldehyde dehydrogenase 1 family, member A3 224959_at 1836 SLC26A2 4.50E-03 1.69 solute carrier family 26 (anion exchanger), member 2 219911_s_at 28231 SLCO4A1 1.35E-02 1.69 solute carrier organic anion transporter family, member 4A1 202238_s_at 4837 NNMT 1.57E-02 1.69 nicotinamide N-methyltransferase 209758_s_at 8076 MFAP5 3.00E-04 1.68 microfibrillar associated protein 5 202679_at 4864 NPC1 9.00E-04 1.68 Niemann-Pick disease, type C1 205399_at 9201 DCLK1 2.00E-03 1.68 doublecortin-like kinase 1 230493_at 387914 SHISA2 2.70E-03 1.68 shisa family member 2 213849_s_at 5521 PPP2R2B 3.10E-03 1.68 protein phosphatase 2, regulatory subunit B, beta 227867_at 129293 TRABD2A 1.50E-02 1.68 TraB domain containing 2A 215034_s_at 4071 TM4SF1 2.07E-02 1.68 transmembrane 4 L six family member 1 203984_s_at 842 CASP9 6.44E-02 1.68 caspase 9 226281_at 92737 DNER 6.89E-02 1.68 delta/notch like EGF repeat containing 201925_s_at 1604 CD55 7.41E-02 1.68 CD55 molecule, decay accelerating factor for complement (Cromer blood group) 203865_s_at 104 ADARB1 9.00E-04 1.67 adenosine deaminase, RNA-specific, B1 216074_x_at 23286 WWC1 4.90E-03 1.67 WW and C2 domain containing 1 219825_at 56603 CYP26B1 6.80E-03 1.67 cytochrome P450, family 26, subfamily B, polypeptide 1 206707_x_at 9750 FAM65B 1.36E-02 1.67 family with sequence similarity 65, member B 228461_at 344558 SH3RF3 1.20E-03 1.66 SH3 domain containing ring finger 3 209676_at 7035 TFPI 2.22E-02 1.66 tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) 214199_at 6441 SFTPD 5.72E-02 1.66 surfactant protein D 209829_at 9750 FAM65B 7.78E-02 1.66 family with sequence similarity 65, member B 206866_at 1002 CDH4 1.20E-03 1.65 cadherin 4, type 1, R-cadherin (retinal) 223125_s_at 81563 C1orf21 2.00E-03 1.65 chromosome 1 open reading frame 21 1555486_a_at 79899 PRR5L 2.90E-03 1.65 proline rich 5 like 213258_at 7035 TFPI 2.90E-03 1.65 tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) 222073_at 1285 COL4A3 3.90E-03 1.65 collagen, type IV, alpha 3 (Goodpasture antigen) 204456_s_at 2619 GAS1 4.60E-03 1.65 growth arrest-specific 1 208108_s_at 554 AVPR2 4.26E-02 1.65 arginine vasopressin receptor 2 215646_s_at 1462 VCAN 7.22E-02 1.65 versican 213085_s_at 23286 WWC1 8.00E-04 1.64 WW and C2 domain containing 1 213988_s_at 6303 SAT1 2.49E-02 1.64 spermidine/spermine N1-acetyltransferase 1 217546_at 4499 MT1M 3.96E-02 1.64 metallothionein 1M 219737_s_at 5101 PCDH9 7.13E-02 1.64 protocadherin 9 224783_at 283991 UBALD2 9.00E-04 1.63 UBA-like domain containing 2 225540_at 4133 MAP2 1.20E-03 1.63 microtubule associated protein 2 202723_s_at 2308 FOXO1 4.30E-03 1.63 forkhead box O1 232206_at 54986 ULK4 3.00E-04 1.62 unc-51 like kinase 4 219714_s_at 55799 CACNA2D3 5.00E-04 1.62 calcium channel, voltage-dependent, alpha 2/delta subunit 3 229585_at 92949 ADAMTSL1 3.40E-03 1.62 ADAMTS like 1 216598_s_at 6347 CCL2 6.70E-03 1.62 chemokine (C-C motif) ligand 2 205795_at 9369 NRXN3 9.80E-03 1.62 neurexin 3 209355_s_at 8613 PLPP3 1.17E-02 1.62 phospholipid phosphatase 3 216944_s_at 3708 ITPR1 3.78E-02 1.62 inositol 1,4,5-trisphosphate receptor, type 1 238419_at 90102 PHLDB2 4.08E-02 1.62 pleckstrin homology-like domain, family B, member 2 206002_at 10149 ADGRG2 7.00E-04 1.61 adhesion G protein-coupled receptor G2 226506_at 79875 THSD4 1.80E-03 1.61 thrombospondin type 1 domain containing 4 36711_at 23764 MAFF 2.14E-02 1.61 v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog F 231067_s_at 9590 AKAP12 2.39E-02 1.61 A kinase (PRKA) anchor protein 12 210775_x_at 842 CASP9 4.69E-02 1.61 caspase 9 229228_at 9586 CREB5 6.56E-02 1.61 cAMP responsive element binding protein 5 225803_at 114907 FBXO32 4.60E-03 1.60 F-box protein 32 207826_s_at 3399 ID3 9.50E-03 1.60 inhibitor of DNA binding 3, dominant negative helix-loop-helix protein 209278_s_at 7980 TFPI2 1.71E-02 1.60 tissue factor pathway inhibitor 2 201508_at 3487 IGFBP4 9.00E-04 1.59 insulin like growth factor binding protein 4 209276_s_at 2745 GLRX 1.80E-03 1.59 glutaredoxin 225989_at 26091 HERC4 3.10E-03 1.59 HECT and RLD domain containing E3 ubiquitin protein ligase 4 229649_at 9369 NRXN3 3.90E-03 1.59 neurexin 3 210592_s_at 6303 SAT1 1.97E-02 1.59 spermidine/spermine N1-acetyltransferase 1 238877_at 2070 EYA4 2.67E-02 1.59 EYA transcriptional coactivator and phosphatase 4 213624_at 10924 SMPDL3A 3.36E-02 1.59 sphingomyelin phosphodiesterase, acid-like 3A 211682_x_at 54490 UGT2B28 4.95E-02 1.59 UDP glucuronosyltransferase 2 family, polypeptide B28 204039_at 1050 CEBPA 5.67E-02 1.59 CCAAT/enhancer binding protein (C/EBP), alpha 204288_s_at 8470 SORBS2 5.74E-02 1.59 sorbin and SH3 domain containing 2 226931_at 83857 TMTC1 7.00E-04 1.58 transmembrane and tetratricopeptide repeat containing 1 209386_at 4071 TM4SF1 1.38E-02 1.58 transmembrane 4 L six family member 1 224480_s_at 84803 GPAT3 2.18E-02 1.58 glycerol-3-phosphate acyltransferase 3 202479_s_at 28951 TRIB2 8.42E-02 1.58 tribbles pseudokinase 2 205074_at 6584 SLC22A5 2.60E-03 1.57 solute carrier family 22 (organic cation/carnitine transporter), member 5 238733_at 4193 MDM2 5.00E-03 1.57 MDM2 proto-oncogene, E3 ubiquitin protein ligase 212226_s_at 8613 PLPP3 8.80E-03 1.57 phospholipid phosphatase 3 221731_x_at 1462 VCAN 1.31E-02 1.57 versican 33304_at 3669 ISG20 1.46E-02 1.57 interferon stimulated exonuclease gene 20kDa 232124_at 729085 FAM198A 2.16E-02 1.57 family with sequence similarity 198, member A 228293_at 91614 DEPDC7 4.93E-02 1.57 DEP domain containing 7 228933_at 4810 NHS 6.40E-02 1.57 Nance-Horan syndrome (congenital cataracts and dental anomalies) 203108_at 9052 GPRC5A 5.00E-04 1.56 G protein-coupled receptor, class C, group 5, member A 230193_at 144406 WDR66 3.20E-03 1.56 WD repeat domain 66 207528_s_at 23657 SLC7A11 6.60E-03 1.56 solute carrier family 7 (anionic amino acid transporter light chain, xc- system), member 11 204698_at 3669 ISG20 1.29E-02 1.56 interferon stimulated exonuclease gene 20kDa 205870_at 624 BDKRB2 4.44E-02 1.56 bradykinin receptor B2 223402_at 54935 DUSP23 1.50E-03 1.55 dual specificity phosphatase 23 202387_at 573 BAG1 1.60E-03 1.55 BCL2-associated athanogene 209087_x_at 4162 MCAM 4.60E-03 1.55 melanoma cell adhesion molecule 242624_at 84448 ABLIM2 8.00E-03 1.55 actin binding LIM protein family, member 2 217623_at 91807 MYLK3 1.30E-02 1.55 myosin light chain kinase 3 202724_s_at 2308 FOXO1 1.57E-02 1.55 forkhead box O1 200730_s_at 7803 PTP4A1 5.37E-02 1.55 protein tyrosine phosphatase type IVA, member 1 210869_s_at 4162 MCAM 2.10E-03 1.54 melanoma cell adhesion molecule 210095_s_at 3486 IGFBP3 2.90E-03 1.54 insulin like growth factor binding protein 3 226431_at 150864 FAM117B 3.70E-03 1.54 family with sequence similarity 117, member B 228625_at 163732 CITED4 1.01E-02 1.54 Cbp/p300-interacting transactivator, with Glu/Asp rich carboxy-terminal domain, 4 202237_at 4837 NNMT 1.36E-02 1.54 nicotinamide N-methyltransferase 228813_at 9759 HDAC4 1.57E-02 1.54 histone deacetylase 4 213790_at 8038 ADAM12 2.61E-02 1.54 ADAM metallopeptidase domain 12 243358_at 3480 IGF1R 2.64E-02 1.54 insulin-like growth factor 1 receptor 213508_at 171546 SPTSSA 4.14E-02 1.54 serine palmitoyltransferase, small subunit A 225496_s_at 54843 SYTL2 5.37E-02 1.54 synaptotagmin-like 2 211475_s_at 573 BAG1 1.40E-03 1.53 BCL2-associated athanogene 202254_at 26037 SIPA1L1 5.70E-03 1.53 signal-induced proliferation-associated 1 like 1 201858_s_at 5552 SRGN 6.80E-03 1.53 serglycin 1555950_a_at 1604 CD55 1.74E-02 1.53 CD55 molecule, decay accelerating factor for complement (Cromer blood group) 227657_at 57484 RNF150 3.90E-02 1.53 ring finger protein 150 229812_at 84196 USP48 4.12E-02 1.53 ubiquitin specific peptidase 48 231015_at 28999 KLF15 7.74E-02 1.53 Kruppel-like factor 15 218113_at 23670 TMEM2 9.95E-02 1.53 transmembrane protein 2 226322_at 83857 TMTC1 1.70E-03 1.52 transmembrane and tetratricopeptide repeat containing 1 219501_at 55068 ENOX1 2.20E-03 1.52 ecto-NOX disulfide-thiol exchanger 1 210015_s_at 4133 MAP2 2.60E-03 1.52 microtubule associated protein 2 208055_s_at 26091 HERC4 2.69E-02 1.52 HECT and RLD domain containing E3 ubiquitin protein ligase 4 226811_at 54855 FAM46C 8.29E-02 1.52 family with sequence similarity 46, member C 203455_s_at 6303 SAT1 9.47E-02 1.52 spermidine/spermine N1-acetyltransferase 1 220227_at 1002 CDH4 1.30E-03 1.51 cadherin 4, type 1, R-cadherin (retinal) 229310_at 114818 KLHL29 1.00E-02 1.51 kelch-like family member 29 210664_s_at 7035 TFPI 2.43E-02 1.51 tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) 209683_at 81553 FAM49A 4.80E-02 1.51 family with sequence similarity 49, member A 228796_at 131034 CPNE4 5.13E-02 1.51 copine IV 205193_at 23764 MAFF 8.39E-02 1.51 v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog F 205462_s_at 3241 HPCAL1 1.30E-03 1.50 hippocalcin-like 1 239352_at 55117 SLC6A15 4.30E-03 1.50 solute carrier family 6 (neutral amino acid transporter), member 15 213280_at 23108 RAP1GAP2 2.18E-02 1.50 RAP1 GTPase activating protein 2 220148_at 64577 ALDH8A1 4.05E-02 1.50 aldehyde dehydrogenase 8 family, member A1 233506_at 9689 BZW1 9.02E-02 1.50 basic leucine zipper and W2 domains 1

Indacaterol (18h) (9 Probe Sets ≡ 9 Gene IDs) Affy Probe Set ID Entrez Gene ID Official Gene Symbol FDR P-Value Fold change UniGene Name Function 237328_at 55195 C14orf105 9.76E-02 3.27 chromosome 14 open reading frame 105 1556420_s_at 388403 YPEL2 4.21E-02 2.24 yippee like 2 216639_at 27286 SRPX2 7.81E-02 2.02 sushi-repeat containing protein, X-linked 2 220006_at 79825 EFCC1 4.21E-02 1.98 EF-hand and coiled-coil domain containing 1 237222_at 123722 FSD2 9.76E-02 1.85 fibronectin type III and SPRY domain containing 2 208335_s_at 2532 ACKR1 3.93E-02 1.59 atypical chemokine receptor 1 (Duffy blood group) 220227_at 1002 CDH4 3.67E-02 1.55 cadherin 4, type 1, R-cadherin (retinal) 1554202_x_at 57010 CABP4 9.76E-02 1.54 calcium binding protein 4 220335_x_at 23491 CES3 3.53E-02 1.53 carboxylesterase 3

Transcripts used for real-time PCR validation are bolded and underlined. *Some genes are recognised by more than one probe set and some probe sets recognise more than one gene. Supplemental TABLE 4. Indacaterol-repressed transcripts with expression level changes <0.67-fold (FDR < 0.10) at 1h, 2h, 6h and 18h.

Indacaterol (2h) (51 Probe Sets ≡ 47 Gene IDs) Affy Probe Set ID* Entrez Gene ID Official Gene Symbol FDR P-Value Fold change UniGene Name Function 232328_at 79818 ZNF552 1.70E-03 0.22 zinc finger protein 552 216345_at 23053 ZSWIM8 5.53E-02 0.34 zinc finger, SWIM-type containing 8 1569262_x_at 90025 UBE3D 6.85E-02 0.36 ubiquitin protein ligase E3D 227404_s_at 1958 EGR1 7.00E-04 0.38 early growth response 1 201009_s_at 10628 TXNIP 7.00E-04 0.40 thioredoxin interacting protein 230673_at 93035 PKHD1L1 9.18E-02 0.40 polycystic kidney and hepatic disease 1 (autosomal recessive)-like 1 201694_s_at 1958 EGR1 3.00E-04 0.40 early growth response 1 201008_s_at 10628 TXNIP 3.00E-04 0.42 thioredoxin interacting protein 1556300_s_at 6492 SIM1 2.20E-03 0.42 single-minded family bHLH transcription factor 1 201010_s_at 10628 TXNIP 3.00E-04 0.45 thioredoxin interacting protein 206876_at 6492 SIM1 5.20E-03 0.45 single-minded family bHLH transcription factor 1 237469_at 7153 TOP2A 2.00E-04 0.46 topoisomerase (DNA) II alpha 244427_at 9493 KIF23 1.50E-03 0.48 kinesin family member 23 1555591_at 80119 PIF1 6.70E-02 0.49 PIF1 5'-to-3' DNA helicase 216109_at 23389 MED13L 1.30E-02 0.51 mediator complex subunit 13-like 1566111_at 9780 PIEZO1 1.20E-03 0.51 piezo-type mechanosensitive ion channel component 1 206115_at 1960 EGR3 5.78E-02 0.52 early growth response 3 1552880_at 89866 SEC16B 2.49E-02 0.53 SEC16 homolog B, endoplasmic reticulum export factor 215175_at 22990 PCNX 1.12E-02 0.53 pecanex homolog (Drosophila) 215508_at 699 BUB1 7.38E-02 0.54 BUB1 mitotic checkpoint serine/threonine kinase 204826_at 899 CCNF 3.40E-03 0.54 cyclin F 240432_x_at 8609 KLF7 1.02E-02 0.54 Kruppel-like factor 7 (ubiquitous) 1553883_at 7652 ZNF99 4.47E-02 0.55 zinc finger protein 99 201693_s_at 1958 EGR1 1.95E-02 0.55 early growth response 1 238049_at 65983 GRAMD3 4.99E-02 0.55 GRAM domain containing 3 232735_at 284615 ANKRD34A 4.00E-03 0.56 ankyrin repeat domain 34A 208025_s_at 8091 HMGA2 1.30E-03 0.56 high mobility group AT-hook 2 232912_at 160897 GPR180 8.34E-02 0.57 G protein-coupled receptor 180 220709_at 80032 ZNF556 7.18E-02 0.57 zinc finger protein 556 23049 NPIPB4 nuclear pore complex interacting protein family, member B4 440345 NPIPB5 nuclear pore complex interacting protein family, member B5 440354 SMG1 SMG1 phosphatidylinositol 3-kinase-related kinase 595101 SMG1P1 SMG1 pseudogene 1 641298 SMG1P2 SMG1 pseudogene 2 244766_at 100132247 SMG1P3 6.15E-02 0.57 SMG1 pseudogene 3 100271836 SMG1P5 SMG1 pseudogene 5 100506060 SMG1P7 SMG1 pseudogene 7 101060596 LOC101060596 serine/threonine-protein kinase SMG1-Like 105369244 LOC105369244 serine/threonine-protein kinase SMG1-like 105369278 LOC105369278 serine/threonine-protein kinase SMG1-Like 219544_at 79866 BORA 2.80E-03 0.59 bora, aurora kinase A activator 228892_at 153769 SH3RF2 9.78E-02 0.60 SH3 domain containing ring finger 2 207286_at 9662 CEP135 2.57E-02 0.61 centrosomal protein 135kDa 227687_at 219844 HYLS1 2.40E-03 0.61 hydrolethalus syndrome 1 201896_s_at 84722 PSRC1 9.00E-04 0.61 proline/serine-rich coiled-coil 1 230462_at 8650 NUMB 3.38E-02 0.62 numb homolog (Drosophila) 218192_at 51447 IP6K2 5.40E-03 0.63 inositol hexakisphosphate kinase 2 238220_at 7403 KDM6A 7.53E-02 0.63 lysine (K)-specific demethylase 6A 1552546_a_at 137994 LETM2 2.06E-02 0.63 leucine zipper-EF-hand containing transmembrane protein 2 235690_at 84622 ZNF594 7.49E-02 0.63 zinc finger protein 594 216061_x_at 5155 PDGFB 1.13E-02 0.63 platelet-derived growth factor beta polypeptide 227801_at 286827 TRIM59 7.00E-03 0.64 tripartite motif containing 59 218726_at 55355 HJURP 9.20E-03 0.65 Holliday junction recognition protein 216081_at 3910 LAMA4 6.14E-02 0.65 laminin, alpha 4 204349_at 9443 MED7 3.98E-02 0.65 mediator complex subunit 7 235165_at 84612 PARD6B 7.48E-02 0.65 par-6 family cell polarity regulator beta 233782_at 10238 DCAF7 1.69E-02 0.65 DDB1 and CUL4 associated factor 7 235476_at 286827 TRIM59 8.20E-03 0.65 tripartite motif containing 59 214918_at 4670 HNRNPM 6.42E-02 0.66 heterogeneous nuclear ribonucleoprotein M 225283_at 91947 ARRDC4 4.50E-03 0.66 arrestin domain containing 4 1552794_a_at 284306 ZNF547 6.66E-02 0.66 zinc finger protein 547

Indacaterol (6h) (51 Probe Sets ≡ 47 Gene IDs) Affy Probe Set ID* Entrez Gene ID Official Gene Symbol FDR P-Value Fold change UniGene Name Function 231600_at 387837 CLEC12B 3.31E-02 0.26 C-type lectin domain family 12, member B 204823_at 89795 NAV3 8.00E-04 0.35 neuron navigator 3 TPSAB1 tryptase alpha/beta 1 207134_x_at 7177 7.04E-02 0.41 64499 TPSB2 tryptase beta 2 (gene/pseudogene) 1552658_a_at 89795 NAV3 7.00E-04 0.43 neuron navigator 3 217262_s_at 9620 CELSR1 6.07E-02 0.46 cadherin, EGF LAG seven-pass G-type receptor 1 223204_at 51313 FAM198B 1.31E-02 0.46 family with sequence similarity 198, member B 223317_at 84266 ALKBH7 4.09E-02 0.47 alkB homolog 7 214978_s_at 8497 PPFIA4 6.39E-02 0.48 protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF), interacting protein (liprin), alpha 4 1552546_a_at 137994 LETM2 1.82E-02 0.48 leucine zipper-EF-hand containing transmembrane protein 2 219872_at 51313 FAM198B 3.00E-04 0.50 family with sequence similarity 198, member B 231053_at 3745 KCNB1 5.67E-02 0.51 potassium channel, voltage gated Shab related subfamily B, member 1 201694_s_at 1958 EGR1 1.74E-02 0.51 early growth response 1 218864_at 7145 TNS1 4.60E-03 0.52 tensin 1 204439_at 10964 IFI44L 1.67E-02 0.52 interferon-induced protein 44-like 227404_s_at 1958 EGR1 1.33E-02 0.53 early growth response 1 202086_at 4599 MX1 7.05E-02 0.53 MX dynamin-like GTPase 1 214453_s_at 10561 IFI44 9.90E-03 0.54 interferon-induced protein 44 206757_at 8654 PDE5A 9.95E-02 0.55 phosphodiesterase 5A, cGMP-specific 211006_s_at 3745 KCNB1 2.14E-02 0.55 potassium channel, voltage gated Shab related subfamily B, member 1 215649_s_at 4598 MVK 1.80E-03 0.55 mevalonate kinase 204363_at 2152 F3 5.74E-02 0.57 coagulation factor III (thromboplastin, tissue factor) 223749_at 114898 C1QTNF2 2.83E-02 0.57 C1q and tumor necrosis factor related protein 2 206553_at 4939 OAS2 8.63E-02 0.57 2'-5'-oligoadenylate synthetase 2 229337_at 9099 USP2 7.04E-02 0.57 ubiquitin specific peptidase 2 1556361_s_at 81573 ANKRD13C 1.51E-02 0.58 ankyrin repeat domain 13C 220381_at 79822 ARHGAP28 7.11E-02 0.58 Rho GTPase activating protein 28 205552_s_at 4938 OAS1 7.12E-02 0.58 2'-5'-oligoadenylate synthetase 1 232666_at 4940 OAS3 4.19E-02 0.60 2'-5'-oligoadenylate synthetase 3 222071_s_at 353189 SLCO4C1 3.53E-02 0.60 solute carrier organic anion transporter family, member 4C1 233234_at 57528 KCTD16 6.71E-02 0.61 potassium channel tetramerization domain containing 16 211520_s_at 2890 GRIA1 6.15E-02 0.62 glutamate receptor, ionotropic, AMPA 1 206825_at 5021 OXTR 1.60E-03 0.62 oxytocin receptor 237767_at 10160 FARP1 5.51E-02 0.62 FERM, ARH/RhoGEF and pleckstrin domain protein 1 233907_s_at 56256 SERTAD4 9.00E-03 0.62 SERTA domain containing 4 235315_at 8848 TSC22D1 6.53E-02 0.62 TSC22 domain family, member 1 204938_s_at 5350 PLN 6.15E-02 0.63 phospholamban 237691_x_at 2027 ENO3 1.51E-02 0.63 enolase 3 (beta, muscle) 204056_s_at 4598 MVK 1.60E-03 0.63 mevalonate kinase 1559060_a_at 96459 FNIP1 5.54E-02 0.63 folliculin interacting protein 1 1554018_at 10457 GPNMB 8.38E-02 0.64 glycoprotein (transmembrane) nmb 240869_at 3745 KCNB1 6.88E-02 0.64 potassium channel, voltage gated Shab related subfamily B, member 1 1559449_a_at 9534 ZNF254 5.51E-02 0.64 zinc finger protein 254 226603_at 219285 SAMD9L 1.40E-02 0.64 sterile alpha motif domain containing 9-like 211018_at 4047 LSS 6.70E-03 0.65 lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase) 211019_s_at 4047 LSS 1.70E-03 0.65 lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase) 227055_at 196410 METTL7B 2.79E-02 0.65 methyltransferase like 7B 235544_x_at 284069 FAM171A2 7.04E-02 0.65 family with sequence similarity 171, member A2 236344_at 5137 PDE1C 1.40E-02 0.65 phosphodiesterase 1C, calmodulin-dependent 70kDa 235760_at 64324 NSD1 4.41E-02 0.66 nuclear receptor binding SET domain protein 1 230708_at 144165 PRICKLE1 2.70E-02 0.66 prickle homolog 1 229767_at 6904 TBCD 1.07E-02 0.67 tubulin folding cofactor D Indacaterol (18h) (1 Probe Set ≡ 1 Gene ID) Affy Probe Set ID* Entrez Gene ID Official Gene Symbol FDR P-Value Fold change UniGene Name Function 240876_x_at 145645 C15orf43 9.51E-02 0.51 chromosome 15 open reading frame 43

Transcripts used for real-time PCR validation are bolded and underlined. *Some genes are recognised by more than one probe set and some probe sets recognise more than one gene. Supplemental TABLE 5. Functional Annotation Clustering of Genes Significantly Induced by Indacaterol in BEAS-2B Cells at 1h, 2h and 6h.

Indacaterol (1h)

Annotation Cluster 1 Enrichment Score: 4.48 Genes Count P -Value Benjamini GOTERM_BP_DIRECT negative regulation of transcription from RNA polymerase II promoter ATF3, BCL6, BHLHE40, BMP2, CITED2, EDN1, EFNA1, ETS2, FEZF2, 23 4.30E-09 5.70E-06 FOXC2, HES1, ID1, ID3, KLF4, MYC, NFIL3, NR4A2, NR4A3, PRDM1, RUNX3, SNAI2, TCF21, TGIF1 GOTERM_MF_DIRECT sequence-specific DNA binding ATF3, BCL6, CSRNP1, ESRRG, ETS2, FOSL2, FOXC1, FOXC2, HES1, 19 1.10E-08 3.40E-06 IRX1, IRX2, KLF2, MAFF, MYC, NFIL3, NR4A1, NR4A2, NR4A3, SNAI2

GOTERM_BP_DIRECT positive regulation of transcription from RNA polymerase II promoter ADRB2, ATF3, BMP2, CEBPD, CITED2, CSRNP1, EDN1, ESRRG, ETS2, 26 1.30E-08 8.40E-06 FEZF2, FOSL2, FOXC1, FOXC2, HES1, IL6, KLF2, KLF4, MAFF, MYC, NR4A1, NR4A2, NR4A3, RGCC, SERPINE1, SOX9, TCF21 GOTERM_MF_DIRECT transcription factor activity, sequence-specific DNA binding ATF3, BCL6, BHLHE40, CEBPD, CITED2, CSRNP1, ESRRG, ETS2, 25 2.00E-08 3.10E-06 FOSL2, FOXC1, FOXC2, HES1, ID1, ID3, KLF2, KLF4, MAFF, MYC, NFIL3, PRDM1, RUNX3, SOX9, TGIF1, TSC22D1, ZFP36L1 GOTERM_MF_DIRECT transcriptional repressor activity, RNA polymerase II core promoter proximal ATF3, BHLHE40, ETS2, HES1, NFIL3, PRDM1, SNAI2, TCF21, TGIF1 9 8.40E-07 8.40E-05 region sequence-specific binding GOTERM_BP_DIRECT transcription from RNA polymerase II promoter ATF3, CEBPD, SRNP1, FEZF2, FOXC1, FOXC2, KLF4, MAFF, MYC, 14 5.90E-05 7.80E-03 NFIL3, RUNX3, SOX9, TCF21, TSC22D1 GOTERM_CC_DIRECT nucleus AKIP1, ANKRD37, ATF3, BCL6, BHLHE40, CEBPD, CITED2, CSNK1A1, 55 1.60E-04 2.00E-02 CSRNP1, DUSP1, DUSP4, DUSP5, ERRFI1, ESRRG, ETS2, FAM71B, FEZF2, FOSL2, FOXC1, FOXC2, HES1, ID1, ID3, IRX1, IRX2, KLF2, KLF4, KLF6, MAFF, MYC, NEDD9, NFIL3, NR4A1, NR4A2, NR4A3, PRDM1, PYHIN1, RBFOX1, RGCC, RGS2, RUNX3, SGK1, SIK1, SNAI2, SOX9, SPIDR, SYNPO2, TCF21, TGIF1, TOB1, TSC22D1, ZFP36L1, ZNF331, ZNF385D, ZNF414 GOTERM_BP_DIRECT transcription, DNA-templated BBCL6, BHLHE40, CEBPD, CITED2, ESRRG, ETS2, FOSL2, HES1, ID1, 20 8.30E-02 6.90E-01 ID3, KLF2, KLF6, NR4A1, NR4A2, NR4A3, PRDM1, SNAI2, TGIF1, ZNF331, ZNF414 GOTERM_CC_DIRECT nucleoplasm AKIP1, ATF3, BCL6, DUSP4, DUSP5, ESRRG, ETS2, FOSL2, FOXC1, 23 1.80E-01 9.40E-01 HES1, ID1, ID3, KLF4, MAFF, MYC, NR4A1, NR4A2, NR4A3, PRDM1 SGK1, SOX9, SPIDR, TGIF1 Annotation Cluster 2 Enrichment Score: 3.49 Genes Count P -Value Benjamini GOTERM_BP_DIRECT cellular response to interleukin-1 CCL20, EDN1, KLF2, HAS2, IL6, MYC, SOX9 7 1.00E-05 2.20E-03 GOTERM_BP_DIRECT cellular response to tumor necrosis factor CCL20, EDN1, KLF2, HAS2, IL6, ZNF36L1 6 1.00E-03 6.30E-02 GOTERM_BP_DIRECT positive regulation of nitric oxide biosynthetic process EDN1, KLF2, KLF4, IL6 4 3.30E-03 1.10E-01 Annotation Cluster 3 Enrichment Score: 3.24 Count P -Value Benjamini GOTERM_BP_DIRECT neural crest cell development EDN1, FOXC1, FOXC2, SNAI2, SOX9 5 1.50E-06 6.70E-04 GOTERM_BP_DIRECT Notch signaling pathway BMP2, FOXC1, FOXC2, HES1, MYC, SNAI2, SOX9 7 1.50E-04 1.50E-02 GOTERM_MF_DIRECT transcription factor activity, RNA polymerase II distal enhancer sequence- BHLHE40, FOXC1, FOXC2, SNAI2, SOX9 5 1.00E-03 3.80E-02 specific binding GOTERM_BP_DIRECT ossification CTGF, FOXC1, FOXC2, RUNX3, SOX9 5 2.40E-03 9.30E-02 GOTERM_MF_DIRECT RNA polymerase II transcription factor activity, sequence-specific DNA binding BHLHE40, FOXC1, RUNX3, SOX9 4 1.10E-01 7.10E-01

Annotation Cluster 4 Enrichment Score: 3.17 Genes Count P -Value Benjamini GOTERM_BP_DIRECT regulation of transcription from RNA polymerase II promoter ATL3, FASL2, MAFF, RUNX3 4 6.00E-01 1.00E+00 Annotation Cluster 5 Enrichment Score: 2.52 Genes Count P -Value Benjamini GOTERM_BP_DIRECT positive regulation of epithelial cell proliferation C5AR1, ID1, IL6, MYC, NR4A3, SOX9 6 6.10E-05 7.30E-03 GOTERM_BP_DIRECT positive regulation of smooth muscle cell proliferation EDN1, IL6, MYC, NR4A3 4 8.50E-03 2.00E-01 Annotation Cluster 6 Enrichment Score: 2.18 Genes Count P -Value Benjamini GOTERM_BP_DIRECT cell-cell signaling ADRB2, AREG, BDNF, BMP2, CCL20, CTGF, EFNA1, EDN1, NTF3 9 4.00E-04 3.30E-02 GOTERM_MF_DIRECT growth factor activity AREG, BDNF, BMP2, CTGF, IL6, NTF3 6 4.90E-03 1.10E-01 GOTERM_MF_DIRECT cytokine activity AREG, EDN1, IL6, BMP2 4 1.20E-01 7.10E-01 Annotation Cluster 7 Enrichment Score: 2.14 Genes Count P -Value Benjamini GOTERM_BP_DIRECT Notch signaling pathway BMP2, FOXC1, FOXC2, HES1, MYC, SNAI2, SOX9 7 1.50E-04 1.50E-02 GOTERM_BP_DIRECT epithelial to mesenchymal transition BMP2, SNAI2, SOX9 3 2.30E-02 3.90E-01 GOTERM_BP_DIRECT negative regulation of canonical Wnt signaling pathway BMP2, CSNK1A1, SNAI2, SOX9 4 1.10E-01 7.50E-01 Annotation Cluster 8 Enrichment Score: 2.12 Genes Count P -Value Benjamini GOTERM_BP_DIRECT cellular response to corticotropin-releasing hormone stimulus NR4A1, NR4A2, NR4A3 3 7.10E-04 4.80E-02 GOTERM_BP_DIRECT fat cell differentiation CEBPD, KLF4, NR4A1, NR4A2, NR4A3 5 1.70E-03 7.60E-02 GOTERM_MF_DIRECT steroid hormone receptor activity ESRRG, NR4A1, NR4A2, NR4A3 4 6.40E-03 1.40E-01 GOTERM_BP_DIRECT steroid hormone mediated signaling pathway ESRRG, NR4A1, NR4A2, NR4A3 4 7.30E-03 1.90E-01 GOTERM_BP_DIRECT transcription initiation from RNA polymerase II promoter CTGF, ESRRG, NR4A1, NR4A2, NR4A3 5 2.20E-02 3.80E-01 GOTERM_MF_DIRECT RNA polymerase II transcription factor activity, ligand-activated sequence- NR4A1, NR4A2, NR4A3 3 2.50E-02 3.30E-01 specific DNA binding GOTERM_BP_DIRECT intracellular receptor signaling pathway NR4A1, NR4A2, NR4A3 3 2.90E-02 4.30E-01 GOTERM_MF_DIRECT zinc ion binding ADAMTS1, ADAMTS15, ESRRG, KLF4, NR4A1, NR4A2, NR4A3, 9 5.40E-01 1.00E+00 ZNF331, ZNF385D Annotation Cluster 9 Enrichment Score: 2.08 Genes Count P -Value Benjamini GOTERM_BP_DIRECT negative regulation of sequence-specific DNA binding transcription factor BHLHE40, ID1, ID3, TRIB1 4 8.50E-03 2.00E-01 activity GOTERM_MF_DIRECT E-box binding BHLHE40, TCF21, MYC 3 2.20E-02 3.10E-01 GOTERM_MF_DIRECT transcription factor binding ID1, ID3, FOXC1, HES1, MYC, TRIB1 6 4.40E-02 4.30E-01 GOTERM_MF_DIRECT protein dimerization activity ID3, MYC, SLC24A2, TRIB1 4 8.10E-02 6.40E-01 Annotation Cluster 10 Enrichment Score: 1.66 Genes Count P -Value Benjamini GOTERM_BP_DIRECT peptidyl-threonine dephosphorylation DUSP1, DUSP4, DUSP5 3 2.50E-03 9.40E-02 GOTERM_BP_DIRECT endoderm formation DUSP1, DUSP4, DUSP5 3 3.00E-03 1.10E-01 GOTERM_MF_DIRECT MAP kinase tyrosine/serine/threonine phosphatase activity DUSP1, DUSP4, DUSP5 3 3.40E-03 8.80E-02 GOTERM_BP_DIRECT inactivation of MAPK activity DUSP1, DUSP4, DUSP5 3 1.30E-02 2.80E-01 GOTERM_BP_DIRECT peptidyl-tyrosine dephosphorylation DUSP1, DUSP4, DUSP5 3 1.50E-01 8.50E-01 GOTERM_MF_DIRECT protein tyrosine phosphatase activity DUSP1, DUSP4, DUSP5 3 1.50E-01 7.70E-01 Annotation Cluster 11 Enrichment Score: 1.51 Genes Count P -Value Benjamini GOTERM_BP_DIRECT positive regulation of ERK1 and ERK2 cascade ACKR3, BMP3, C5AR1, CCL20, CTGF, IL6, SPRY2 7 1.40E-03 7.00E-02 GOTERM_BP_DIRECT response to lipopolysaccharide C5AR1, CXCL2, CXCL3, EDN1, IL12RB2, TRIB1 6 5.80E-03 1.60E-01 GOTERM_BP_DIRECT positive regulation of neutrophil chemotaxis C5AR1, CXCL2, CXCL3 3 1.00E-02 2.30E-01 GOTERM_BP_DIRECT chemokine-mediated signaling pathway ACKR3, CCL20, CXCL2, CXCL3 4 1.30E-02 2.80E-01 GOTERM_MF_DIRECT chemokine activity CCL20, CXCL2, CXCL3 3 4.30E-02 4.40E-01 GOTERM_BP_DIRECT inflammatory response BCL6, BMP2, C5AR1, CCL20, CXCL2, CXCL3, IL6, 7 4.90E-02 5.60E-01 GOTERM_BP_DIRECT chemotaxis ACKR3, C5AR1, CXCL2, CCL20, 4 5.40E-02 5.80E-01 GOTERM_BP_DIRECT immune response C5AR1, CCL20, CXCL2, CXCL3, IL6, NFIL3, SH2D6 7 7.40E-02 6.60E-01 GOTERM_BP_DIRECT cell chemotaxis C5AR1, CCL20, CXCL2 3 7.50E-02 6.60E-01 GOTERM_BP_DIRECT G-protein coupled receptor signaling pathway ACKR3, AREG, CCL20, CXCL2, CXCL3, EDN1 6 7.60E-01 1.00E+00 Indacaterol (2h) Annotation Cluster 1 Enrichment Score: 7.64 Genes Count P -Value Benjamini UP_SEQ_FEATURE DNA-binding region:Basic motif ATF3, BHLHE40, CEBPA, CEBPD, CREB3L2, EPAS1, FOS, FOSB, 17 4.90E-09 4.60E-06 FOSL2, HES1, JUNB, MAFF, MAFK, MYC, NFIL3, PHTF2, TCF21 Annotation Cluster 2 Enrichment Score: 6.81 Genes Count P -Value Benjamini GOTERM_BP_DIRECT positive regulation of transcription from RNA polymerase II promoter ATF3, ATXN7, BMP2, CEBPA, CEBPD, CITED4, CREB3L2, EDN1, 53 3.20E-13 7.50E-10 EPAS1, ETS2, FGFR2, FOS, FOSB, FOSL2, FOXC1, FOXC2, FOXO1, FSTL3, GREM1, HES1, HOXA5, IL6, IL11, INHBA, JAG1, JUNB, KLF4, LPIN1, MAFF, MAFK, MEF2D, MYC, NFATC1, NOG, NR4A1, NR4A2, NR4A3, PPARGC1A, PROX2, REL, RGCC, SERPINE1, SIX2, SMAD2, SOX4, SOX9, TCF21, TFAP2A, TFAP2C, VGLL2, WNT5A, ZBTB38, ZNF462 GOTERM_MF_DIRECT transcriptional activator activity, RNA polymerase II core promoter proximal CEBPA, CEBPD, CREB3L2, EPAS1, FOS, FOSB, FOXC2, HOXA5, JUNB, 22 8.60E-10 4.40E-07 region sequence-specific binding KLF4, MEF2D, MYC, NR4A1, NR4A2, NR4A3, SIX2, SMAD2, SOX4, SOX9, TCF21, TFAP2A, TFAP2C GOTERM_BP_DIRECT transcription from RNA polymerase II promoter ATF3, CEBPA, CEBPD, CREB3L2, CTDP1, EPAS1, FOS, FOSB, FOXC1, 32 1.40E-09 1.10E-06 FOXC2, HIVEP1, HIVEP2, HIVEP3, HOXA5, JUNB, KLF4, MAFF, MAFK, MEF2D, MYC, NFATC1, NFIL3, REL, RUNX3, SIX2, SMAD2, SOX4, SOX9, TCF21, TFAP2A, TFAP2C, TSC22D1 GOTERM_MF_DIRECT transcription factor activity, sequence-specific DNA binding ATF3, BCL6, BHLHE40, BNC1, CEBPA, CEBPD, CREB3L2, EPAS1, ETS2, 43 2.50E-08 4.30E-06 FOS, FOSB, FOSL2, FOXC1, FOXC2, HES1, HIVEP2, HIVEP3, HOXA5, ID3, KLF3, KLF4, KLF9, MAFF, MAFK, MEF2D, MYC, NFATC1, NFIL3, NR2F2, PRDM1, REL, RUNX3, SIX2, SMAD2, SOX4, SOX9, TBX3, TFAP2A, TFAP2C, TGIF1, TSC22D1, WNT5A, ZBTB38

GOTERM_CC_DIRECT nucleus ANKRD37, AREG, ARID5B, ATF3, ATXN7, BAG1, BCL6, BCOR, 123 2.90E-06 7.10E-04 BHLHE40, BNC1, BTG1, C8orf4, C9orf72, C15orf48, CASP9, CDKN1C, CEBPA, CEBPD, CITED4, CPEB4, CREB3L2, CSNK1E, CTDP1, DGKD, DLC1, DUSP1, DUSP4, EPAS1, ERRFI1, ETS2, FERMT2, FGFR2, FILIP1L, FOS, FOSB, FOSL2, FOXC1, FOXC2, FOXO1, FSTL3, GDF15, GEM, HERC4, HES1, HIVEP1, HIVEP2, HIVEP3, HLX, HOXA5, ID3, IGFBP3, ING1, IRF2BP2, IRX1, IRX2, IRX4, ISG20, JMY, JUP, KLF3, KLF6, KLF9, LATS2, LBH, LMCD1, LPIN1, MAFF, MAFK, MBP, MEF2D, MYC, NEDD9, NFATC1, NFIL3, NFKBIZ, NR2F2, NR4A1, NR4A2, NR4A3, PHTF2, PIP5K1A, PMAIP1, PPARGC1A, PPM1D, PRDM1, PRKCE, PROX2, PTP4A1, REL, RGCC, RGS2, RHOB, RUNX3, SGK1, SH3BP4, SIK1, SIK2, SIX2, SLC2A14, SMAD2, SNAI2, SNRK, SOX4, SOX9, SPRY2, STC1, TBX3, TCF21, TFAP2A, TFAP2C, TGIF1, TLE1, TLE3, TNFAIP3, TRIB1, TSC22D1, USP36, VGLL2, WWC1, ZBTB38, ZNF331, ZNF462, ZNF503 GOTERM_CC_DIRECT nucleoplasm A1CF, ARID5B, ATF3, ATXN7, BCL6, BNC1, C9orf72, CREB3L2, 62 4.30E-03 1.40E-01 CSNK1E, CTDP1, DUSP4, DUSP5, ELL2, EPAS1, ETS2, FERMT2, FGFR2, FOS, FOSL2, FOXC1, FOXO1, FSTL3, HES1, HIVEP2, ID3, IL15, ING1, ISG20, JMY, JUNB, KLF4, KLF9, LMCD1, LPIN1, MAFF, MAFK, MCTP2, MEF2D, MYC, NFATC1, NR4A1, NR4A2, NR4A3, PFKFB3, PIP5K1A, PPARGC1A, PRDM1, REL, SAP30, SGK1, SIX2, SMAD2, SOX4, SOX9, SPIDR, TFAP2A, TFAP2C, TGIF1, TLE1, TLE3, USP2, USP48 GOTERM_BP_DIRECT transcription, DNA-templated ARID5B, ATXN7, BCL6, BCOR, BHLHE40, BNC1, CEBPA, CEBPD, 49 7.50E-03 1.80E-01 CITED4, EPAS1, ERN1, ETS2, FOSL2, FOXO1, FSTL3, HES1, HLX, ID3, IRF2BP2, KLF3, KLF6, KLF9, LBH, LMCD1, LPIN1, MAFK, MEF2D, MN1, NFKBIZ, NR2F2, NR4A1, NR4A2, NR4A3, PHTF2, PRDM1, PROX2, SAP30, SMAD2, SNAI2, TBX3, TGIF1, TLE1, TLE3, VGLL2, WWC1, ZBTB38, ZNF331, ZNF462, ZNF503 Annotation Cluster 3 Enrichment Score: 3.73 Genes Count P -Value Benjamini GOTERM_BP_DIRECT neural crest cell development EDN1, FOXC1, FOXC2, SNAI2, SOX9 5 5.50E-05 7.40E-03 GOTERM_MF_DIRECT transcription factor activity, RNA polymerase II distal enhancer sequence- BHLHE40, CEBPA, FOXC1, FOXC2, NFATC1, SNAI2, SOX9, VGLL2 8 1.40E-04 7.00E-03 specific binding GOTERM_BP_DIRECT Notch signaling pathway BMP2, CEBPA, FOXC1, FOXC2, HES1, JAG1, MYC, SNAI2, SOX9 9 8.60E-04 3.80E-02 Annotation Cluster 4 Enrichment Score: 3.41 Genes Count P -Value Benjamini GOTERM_MF_DIRECT growth factor activity AREG, BDNF, BMP2, CTGF, DKK1, FGF9, DF15, HBEGF, IL6, IL11, 14 4.80E-06 4.90E-04 INHBA, JAG1, NTF3, VEGFC GOTERM_BP_DIRECT cell-cell signaling AREG, BDNF, BMP2, CCL20, CTGF, EDN1, EFNA1, FGF9, FGFR2, 16 3.70E-05 5.70E-03 GDF15, GREM1, IL11, IL15, INHBA, NTF3, TFAP2C GOTERM_MF_DIRECT cytokine activity AREG, BMP2, EDN1, GDF15, GREM1, IL6, IL11, IL15, INHBA, WNT5A 10 3.50E-03 1.10E-01

Annotation Cluster 5 Enrichment Score: 3.34 Genes Count P -Value Benjamini GOTERM_BP_DIRECT cell fate commitment BMP2, FGFR2, GAS1, PRDM1, SMAD2, SPRY2, WNT4, WNT5A 8 1.30E-05 2.90E-03 GOTERM_BP_DIRECT epithelial to mesenchymal transition BMP2, FGFR2, NOG, SNAI2, SOX9, WNT4, WNT5A 7 2.30E-05 4.00E-03 Annotation Cluster 6 Enrichment Score: 2.16 Genes Count P -Value Benjamini GOTERM_CC_DIRECT integral component of plasma membrane BDKRB2, C5AR1, CD200, CD55, CLDN1, DLCK1, EFNA1, FGFR2, 45 1.20E-05 1.40E-03 GPRC5A, GPR37, HBEGF, HLA-DQA1, HS6ST1, IGF1R, IL15, IL1RAP, JAG1, KCNJ2, KCNK1, KCNK15, NPC1, PAG1, PLAUR, PLPP3, PLXNA2, RAPGEF2, SCARA5, SEMA6D, SLC2A3, SLC7A1, SLC7A2, SLC7A5, SLC7A11, SLC16A6, SLC19A2, SLC22A4, SLC22A5, SLC26A2, SLC38A2, SLCO4A1, STEAP4, TFRC, THBD, TRABD2A, TM4SF1

GOTERM_CC_DIRECT extracellular space ADAMTS15, AREG, B4GALT1, BMP2, C9orf72, CCL2, CCL20, CES3, 41 8.30E-05 6.70E-03 CTGF, CXCL2, DKK1, DMBT1, EDN1, FGF9, FSTL3, GDF15, GREM1, HBEGF, IGFBP3, IGFBP4, IL6, IL6R, IL11, IL15, KIT, LMCD1, LPL, LYPD3, METRNL, NOG, PAPPA, SERPINE1, SMPDL3A, STC1, STC2, TFRC, THBD, ULBP2, VEGFC, WNT4, WNT5A

GOTERM_CC_DIRECT extracellular region BDNF, BMP2, CCL2, CCL20, CD55, CHSY1, CRISPLD2, CTGF, CXCL2, 39 8.20E-03 2.00E-01 DKK1, DMBT1, EDN1, EFNA1, FGF9, FGFR2, FSTL3, GDF15, HBEGF, IGFBP3, IGFBP4, IL6, IL6R, IL11, IL15, IL1RAP, INHBA, JAG1, LPL, MFAP5, NOG, NPC1, NTF3, PAPPA, SERPINE1, STC2, TFRC, VEGFC, WNT4, WNT5A GOTERM_CC_DIRECT integral component of membrane ACKR3, AREG, B4GALT1, BDKRB2, BDNF, C15orf48, CD200, CD274, 70 9.60E-01 1.00E+00 CHSY1, CLDN1, CREB3L2, EVA1C, FAM87A, FGFR2, FLVCR2, GALNT15, GAS1, GCNT2, GPAT3, GPRC5A, HLA-DQA1, HRK, HS3ST1, HS6ST1, IGF1R, IL1RAP, IL6R, JAG1, KCNK1, KIT, LRRC8A, LYPD3, MARC1, MCTP2, MEGF9, NPC1, PAPPA, PCDH9, PHLDB2, PHTF2, PLAUR, RNF19A, SEMA6D, SHISA2, SIRPA, SLC2A3, SLC2A14, SLC7A1, SLC7A5, SLC7A11, SLC10A7, SLC16A6, SLC16A14, SLC19A2, SLC22A4, SLC22A5, SLC26A2, SLC30A1, SLC38A2, SLCO4A1, SMAD2, ST3GAL1, ST3GAL4, ST3GAL6, STEAP4, SUSD6, TFRC, TM4SF1, TMEM2, WNT5A Annotation Cluster 7 Enrichment Score: 2.05 Count P -Value Benjamini GOTERM_BP_DIRECT amino acid transport MYC, SLC7A1, SLC7A2, SLC7A5, SLC7A11, SLC38A2 6 3.20E-04 2.10E-02 GOTERM_MF_DIRECT amino acid transmembrane transporter activity SLC7A1, SLC7A2, SLC7A5, SLC7A11, SLC38A2 5 8.60E-03 2.30E-01 GOTERM_MF_DIRECT antiporter activity SLC7A1, SLC7A2, SLC7A5 3 5.00E-02 5.30E-01 GOTERM_BP_DIRECT transport ATP1B3, NPTX1, SLC7A1, SLC7A2, SLC7A5, SLC19A2, 6 7.20E-01 1.00E+00 Annotation Cluster 8 Enrichment Score: 1.98 Genes Count P -Value Benjamini KEGG_PATHWAY TNF signaling pathway CCL2, CCL20, CREB3L2, CXCL2, EDN1, FOS, IL6, IL15, JUNB, MAP3K8, 12 1.80E-05 3.40E-03 SOCS3, TNFAIP3 KEGG_PATHWAY Rheumatoid arthritis CCL2, CCL20, FOS, IL6, IL11, IL15, HLA-DQA1 7 1.20E-02 2.00E-01 Annotation Cluster 9 Enrichment Score: 1.80 Genes Count P -Value Benjamini GOTERM_BP_DIRECT neutrophil chemotaxis C5AR1, CCL2, CCL20, EDN1, PDE4B, PDE4D 6 5.60E-03 1.50E-01 GOTERM_BP_DIRECT cellular response to interleukin-1 CCL2, CCL20, END1, IL6, MYC, SOX9 6 7.60E-03 1.80E-01 GOTERM_BP_DIRECT response to amino acid CCL2, CTGF, EDN1, IL6 4 1.60E-02 2.80E-01 GOTERM_BP_DIRECT cellular response to interferon-gamma CCL2, CCL20, EDN1, MYC, WNT5A 5 1.70E-02 2.90E-01 GOTERM_BP_DIRECT cellular response to drug CCL2, EDN1, MYC, PDE4B, TFRC 5 3.10E-02 4.10E-01 GOTERM_BP_DIRECT cellular response to tumor necrosis factor CEBPA, CCL2, CCL20, EDN1, IL6, PPARGC1A 6 4.20E-02 4.80E-01 Annotation Cluster 10 Enrichment Score: 1.75 Genes Count P -Value Benjamini INTERPRO Major facilitator superfamily domain FLVCR2, SLC2A3, SLC2A14, SLC16A6, SLC16A14, SLC19A2, SLC22A4, 9 1.70E-03 1.80E-01 SLC22A5, SLCO4A1 GOTERM_MF_DIRECT transmembrane transporter activity SLC2A3, SLC2A14, SLC22A5 3 2.10E-01 8.40E-01 Annotation Cluster 11 Enrichment Score: 1.52 Genes Count P -Value Benjamini GOTERM_BP_DIRECT cellular response to corticotropin-releasing hormone stimulus NR4A1, NR4A2, NR4A3 3 4.30E-03 1.30E-01 GOTERM_MF_DIRECT RNA polymerase II transcription factor activity, ligand-activated sequence- NR2F2, NR4A1, NR4A2, NR4A3 4 2.40E-02 3.90E-01 specific DNA binding GOTERM_BP_DIRECT intracellular receptor signaling pathway NR2F2, NR4A1, NR4A2, NR4A3 4 2.80E-02 3.80E-01 GOTERM_MF_DIRECT steroid hormone receptor activity NR2F2, NR4A1, NR4A2, NR4A3 4 7.20E-02 5.90E-01 GOTERM_BP_DIRECT steroid hormone mediated signaling pathway NR2F2, NR4A1, NR4A2, NR4A3 4 7.60E-02 6.50E-01 GOTERM_BP_DIRECT transcription initiation from RNA polymerase II promoter CTGF, NR4A1, NR4A2, NR4A3, PPARGC1A 5 2.70E-01 9.50E-01 Annotation Cluster 12 Enrichment Score: 1.52 Genes Count P -Value Benjamini GOTERM_BP_DIRECT regulation of cell growth CTGF, IGFBP3, IGFBP4, SGK1, SOCS2, TFRC 6 1.20E-02 2.40E-01 Annotation Cluster 13 Enrichment Score: 1.52 Genes Count P -Value Benjamini GOTERM_CC_DIRECT anchored component of plasma membrane EFNA1, GAS1, LYPD3, ULBP2 4 9.60E-03 2.10E-01 Annotation Cluster 14 Enrichment Score: 1.52 Genes Count P -Value Benjamini GOTERM_BP_DIRECT monocyte chemotaxis CCL2, CCL20, IL6, IL6R 4 3.60E-02 4.40E-01 Annotation Cluster 15 Enrichment Score: 1.49 Genes Count P -Value Benjamini GOTERM_BP_DIRECT cellular response to transforming growth factor beta stimulus EDN1, PPARGC1A, SOX9, WNT4, WNT5A 5 1.00E-02 2.10E-01 GOTERM_BP_DIRECT cartilage development CREB3L2, EDN1, NOG, SOX9, WNT5A 5 1.90E-02 3.10E-01 Annotation Cluster 16 Enrichment Score: 1.47 Genes Count P -Value Benjamini GOTERM_BP_DIRECT protein phosphorylation BMP2, CSNK1E, CCL2, DCLK1, ERN1, EIF2AK3, IGFBP3, LATS2, 20 3.80E-04 2.40E-02 MAP3K8, PLK2, PRKCE, PDK4, RUNX3, SIK1, SIK2, SGK1, SMAD2, SNRK, ST3GAL1, TRIB1 GOTERM_BP_DIRECT peptidyl-serine phosphorylation CSNK1E, DCLK1, EIF2AK3, LATS2, PLK2, PRKCE, SGK1 7 2.10E-02 3.40E-01 GOTERM_MF_DIRECT protein serine/threonine kinase activity CSNK1E, DCLK1, EIF2AK3, ERN1, LATS2, MAP3K8, PLK2, PRKCE, 12 6.20E-02 5.70E-01 SGK1, SIK1, SIK2, SNRK GOTERM_MF_DIRECT protein kinase activity CCL2, CSNK1E, DCLK1, EIF2AK3, MAP3K8, PDK4, PRKCE, SGK223, 11 9.30E-02 6.30E-01 SNRK, SOX9, TRIB1 GOTERM_MF_DIRECT magnesium ion binding ERN1, GEM, MAP3K8, SIK1, SIK2, SNRK 6 2.80E-01 8.90E-01 GOTERM_MF_DIRECT ATP binding CSNK1E, DCLK1, DGKD, EIF2AK3, ERN1, FGFR2, IGF1R, KIT, LATS2, 26 5.90E-01 1.00E+00 MAP3K8, PDK4, PFKFB3, PIP5K1A, PLK2, PRKCE, RHOBTB3, RIMKLB, RUNX3, SGK1, SGK223, SIK1, SIK2, SLC22A4, SLC22A5, SNRK, TRIB1

Annotation Cluster 17 Enrichment Score: 1.43 Count P -Value Benjamini GOTERM_BP_DIRECT cellular response to dexamethasone stimulus CCL2, CASP9, ERRFI1, FOXO1, IL6 5 1.50E-03 5.70E-02 GOTERM_BP_DIRECT response to antibiotic CCL2, CASP9, IL6 3 1.10E-01 7.30E-01 Annotation Cluster 18 Enrichment Score: 1.42 Genes Count P -Value Benjamini GOTERM_BP_DIRECT endoderm formation DKK, DUSP1, DUSP4, DUSP5, NOG, SMAD2 6 1.10E-06 3.50E-04 GOTERM_BP_DIRECT peptidyl-threonine dephosphorylation DUSP1, DUSP4, DUSP5, PPM1D 4 7.60E-04 3.50E-02 GOTERM_MF_DIRECT MAP kinase tyrosine/serine/threonine phosphatase activity DKK, DUSP1, DUSP4, DUSP5, NOG, SMAD2 3 2.00E-02 3.80E-01 GOTERM_BP_DIRECT inactivation of MAPK activity DUSP1, DUSP4, DUSP5 3 6.90E-02 6.20E-01 GOTERM_MF_DIRECT phosphatase activity DUSP4, DUSP5, PTP4A1 3 1.50E-01 7.60E-01 GOTERM_BP_DIRECT peptidyl-tyrosine dephosphorylation DUSP1, DUSP4, DUSP5, PTP4A1 4 2.40E-01 9.30E-01 GOTERM_MF_DIRECT protein tyrosine phosphatase activity DUSP1, DUSP4, DUSP5, PTP4A1 4 2.50E-01 8.80E-01 Annotation Cluster 19 Enrichment Score: 1.37 Genes Count P -Value Benjamini GOTERM_MF_DIRECT symporter activity SLC10A7, SLC16A6, SLC16A14, SLC22A4, SLC22A5, SLC38A2 6 1.80E-03 7.40E-02 GOTERM_BP_DIRECT sodium ion transport ATP13B, SGK1, SLC10A7, SLC22A4, SLC22A5, SLC38A2 6 1.30E-02 2.40E-01 GOTERM_BP_DIRECT transmembrane transport FLVCR2, SLC10A7, SLC16A6, SLC16A14 4 8.00E-01 1.00E+00 Annotation Cluster 20 Enrichment Score: 1.31 Genes Count P -Value Benjamini UP_SEQ_FEATURE DNA-binding region:Homeobox; TALE-type IRX1, IRX2, IRX4, TGIF1 4 3.40E-03 3.30E-01 INTERPRO Iroquois-class homeodomain protein IRX1, IRX2, IRX4 3 3.70E-03 3.00E-01

Indacaterol (6h)

Annotation Cluster 1 Enrichment Score: 4.86 Genes Count P -Value Benjamini GOTERM_CC_DIRECT extracellular space ALB, B4GALT1, C1QTNF6, CCL2, CD59, CES3, CMTM6, DEFB1, 27 1.20E-03 5.50E-02 DMBT1, ENOX1, GDF15, GPC3, IGFBP3, IGFBP4, IL20, KRT86, LPL, MCAM, MSMB, PAPPA, SFTPD, SMPDL3A, SRGN, TFPI, VCAN, WNT4, WNT5A GOTERM_CC_DIRECT extracellular region ADAM12, ALB, CCL2, CD55, COL4A3, CRISPLD1, CRISPLD2, DEFB1, 27 1.20E-02 2.60E-01 DMBT1, ESR2, FAM198A, FGFR2, GDF15, IGFBP3, IGFBP4, IL20, LPL, MFAP5, NETO1, NPC1, PAPPA, SFTPD, SRGN, TFPI, VCAN, WNT4, WNT5A Annotation Cluster 2 Enrichment Score: 3.77 Genes Count P -Value Benjamini GOTERM_CC_DIRECT integral component of plasma membrane ADGRG2, AVPR2, BDKRB2, C5AR1, CD200, CD55, CDH4, CNR1, 32 3.70E-05 7.30E-03 DCLK1, EPHA5, FGFR2, GPC3, GPRC5A, IGF1R, KCNK15, LPAR6, NPC1, NRXN3, PLPP3, PTPRO, SLC6A15, SLC7A2, SLC7A11, SLC16A6, SLC22A3, SLC22A4, SLC22A5, SLC26A2, STEAP4, TM4SF1, GOTERM_CC_DIRECT plasma membrane ACKR1, ADAM12, ADAM22, ADGRD1, AKAP12, ANTXR1, ATP1B3, 62 7.60E-04 7.20E-02 AVPR2, B4GALT1, BDKRB2, C5AR1, CACNA2D3, CD55, CD59, CD200, CDH4, CFTR, CLMP, CNR1, COBL, DNER, DOCK4, ENOX1, EPHA5, FGFR2, GAS1, GNAL, GPC3, GPRC5A, GUCY1A2, IGF1R, ITPR1, JUP, KCNG1, KCNK15, LPAR6, LPL, MCAM, MDM2, NFASC, NOS3, PCDH9, PHLDB2, PLPP3, PTPRO, RAP1GAP2, RGS2, SIRPA, SLC6A15, SLC7A2, SLC7A11, SLC16A14, SLC22A3, SLC22A4, SLC22A5, SLC26A2, SLCO4A1, STEAP4, SYTL2, TFPI, WNT4, WNT5A

GOTERM_CC_DIRECT integral component of membrane ACKR1, ADAM12, ADAM22, ADGRD1, ADGRG2, ANTXR1, AVPR2, 64 5.10E-02 5.20E-01 B4GALT1, BDKRB2, C15orf48, C1QTNF6, CD200, CDH4, CFTR, CLMP, CMTM6, CNR1, DNER, EPHA5, FGFR2, GALNT15, GAS1, GPAT3, GPRC5A, GRAMD1B, HRK, IGF1R, ITPR1, LPAR6, MARC1, MCAM, NETO1, NFASC, NPC1, NRXN3, PAPPA, PCDH9, PDE3A, PHLDB2, PLD5, PLXDC2, PTPRO, RNF150, SHISA2, SIRPA, SLC6A15, SLC7A11, SLC16A6, SLC16A14, SLC22A3, SLC22A4, SLC22A5, SLC26A2, SLCO4A1, SPTLC3, SPTSSA, STEAP4, TM4SF1, TMEM2, TMTC1, UGT1A8, UGT2B28, UST, WNT5A Annotation Cluster 3 Enrichment Score: 2.10 Genes Count P -Value Benjamini GOTERM_BP_DIRECT cell fate commitment FGFR2, GAS1, HEY1, PRDM1, WNT4, WNT5A 6 1.10E-04 7.70E-02 GOTERM_BP_DIRECT epithelial to mesenchymal transition FGFR2, WNT4, WNT5A 3 4.90E-02 9.10E-01 Annotation Cluster 4 Enrichment Score: 2.10 Genes Count P -Value Benjamini GOTERM_BP_DIRECT cAMP catabolic process PDE3A, PDE4D, PDE7B, PDE10A 4 4.60E-04 2.00E-01 GOTERM_MF_DIRECT 3',5'-cyclic-AMP phosphodiesterase activity PDE3A, PDE4D, PDE7B, PDE10A 4 4.90E-04 1.80E-01 GOTERM_MF_DIRECT 3',5'-cyclic-nucleotide phosphodiesterase activity PDE3A, PDE4D, PDE10A 3 2.50E-02 7.60E-01 GOTERM_MF_DIRECT cAMP binding PDE3A, PDE4D, PDE10A 3 2.70E-02 7.40E-01 Annotation Cluster 5 Enrichment Score: 2.08 Genes Count P -Value Benjamini GOTERM_BP_DIRECT quaternary ammonium group transport SLC22A3, SLC22A4, SLC22A5 3 1.10E-03 3.10E-01 GOTERM_MF_DIRECT quaternary ammonium group transmembrane transporter activity SLC22A3, SLC22A4, SLC22A5 3 1.10E-03 2.00E-01 Annotation Cluster 6 Enrichment Score: 2.02 Genes Count P -Value Benjamini GOTERM_CC_DIRECT proteinaceous extracellular matrix ADAMTSL1, COL4A3, CRISPLD2, GPC3, SFTPD, SMOC1, TFPI2, 10 1.70E-03 6.50E-02 VCAN, WNT4, WNT5A GOTERM_CC_DIRECT endoplasmic reticulum lumen ADAMTSL1, CES3, COL4A3, WNT4, WNT5A 5 1.30E-01 7.70E-01 Annotation Cluster 7 Enrichment Score: 1.73 Genes Count P -Value Benjamini GOTERM_MF_DIRECT symporter activity SLC16A6, SLC16A14, SLC22A4, SLC22A5 4 1.70E-02 6.80E-01 Annotation Cluster 8 Enrichment Score: 1.56 Genes Count P -Value Benjamini KEGG_PATHWAY Pathways in cancer BDKRB2, CASP9, CEBPA, COL4A3, FGFR2, FOXO1, IGF1R, JUP, 12 7.60E-03 4.80E-01 LPAR6, MDM2, WNT4, WNT5A

Indacaterol-induced genes (defined by using Affymetrix probe sets) with significant expression level changes of >1.5-fold (based on a FDR P -value of <0.1) were subjected to functional annotation clustering using DAVID (default parameters). Clusters with enrichment scores >1.3 are shown. Supplemental TABLE 6. Functional Annotation Clustering of Genes Significantly Repressed by Indacaterol in BEAS-2B Cells at 1h, 2h and 6h.

Indacaterol (1h) Annotation Cluster 1 Enrichment Score: 1.35 Genes Count P -Value Benjamini GOTERM_BP_DIRECT DNA repair CHEK1, EID3, RECQL5 3 5.40E-02 8.90E-01

Indacaterol (2h) Annotation Cluster 1 Enrichment Score: 2.72 Genes Count P -Value Benjamini GOTERM_BP_DIRECT cell division BUB1, BORA, CCNF, HMGA2, PARD6B, PSRC1 6 1.20E-03 3.20E-01 GOTERM_BP_DIRECT mitotic nuclear division BUB1, BORA, CCNF, HMGA2 4 2.00E-02 7.80E-01 GOTERM_CC_DIRECT cytosol BORA, BUB1, CEP135, KIF23, PARD6B, PKHD1L1, PSRC1, SEC16B, TXNIP 9 5.30E-01 1.00E+00 Annotation Cluster 2 Enrichment Score: 1.91 Genes Count P -Value Benjamini GOTERM_MF_DIRECT DNA binding EGR1, EGR3, HJURP, HMGA2, KLF7, SIM1, TOP2A, ZNF99, ZNF547, ZNF556, 12 1.40E-03 1.30E-01 ZNF594, ZNF738 GOTERM_BP_DIRECT transcription, DNA-templated EGR3, KLF7, MED13L, SIM1, TXNIP, ZNF99, ZNF547, ZNF552, ZNF556, ZNF594, 11 1.20E-02 7.10E-01 ZNF738 GOTERM_CC_DIRECT nucleus CCNF, EGR1, EGR3, HJURP, HMGA2, HYLS1, IP6K2, KDM6A, KIF23, KLF7, 21 1.50E-02 7.20E-01 NUMB, PARD6B, SIM1, TOP2A, TXNIP, ZNF99, ZNF547, ZNF552, ZNF556, ZNF594, ZNF738 GOTERM_MF_DIRECT nucleic acid binding KLF7, ZNF99, ZNF552, ZNF547, ZNF556, ZNF594, ZNF738 7 2.70E-02 7.70E-01 GOTERM_BP_DIRECT regulation of transcription, DNA-templated EGR3, HMGA2, ZNF99, ZNF552, ZNF547, ZNF556, ZNF594, ZNF738 8 5.60E-02 8.90E-01 GOTERM_CC_DIRECT intracellular KIF23, TRIM59, ZNF99, ZNF547, ZNF552, ZNF556, ZNF738 7 9.10E-02 8.10E-01 GOTERM_MF_DIRECT metal ion binding EGR1, EGR3, KDM6A, KLF7, ZNF99, ZNF547, ZNF552, ZNF556, ZNF594 9 1.10E-01 9.80E-01 GOTERM_MF_DIRECT transcription factor activity, sequence-specific DNA binding EGR1, EGR3, KLF7, SIM1, ZNF552 5 1.90E-01 1.00E+00 GOTERM_MF_DIRECT zinc ion binding EGR1, KLF7, SH3RF2, TRIM59, ZSWIM8 5 3.00E-01 1.00E+00

Indacaterol (6h)

Annotation Cluster 1 Enrichment Score: 2.31 Genes Count P -Value Benjamini GOTERM_BP_DIRECT type I interferon signaling pathway EGR1, MX1, OAS1, OAS2, OAS3 5 1.30E-05 3.70E-03 GOTERM_MF_DIRECT 2'-5'-oligoadenylate synthetase activity OAS1, OAS2, OAS3 3 2.80E-05 2.70E-03 GOTERM_BP_DIRECT response to virus IFI44, MX1, OAS1, OAS2, OAS3 5 1.10E-04 1.60E-02 GOTERM_BP_DIRECT defense response to virus IFI44L, MX1, OAS1, OAS2, OAS3 5 5.10E-04 4.90E-02 GOTERM_MF_DIRECT nucleotidyltransferase activity OAS1, OAS2, OAS3 3 1.60E-03 7.50E-02 GOTERM_BP_DIRECT negative regulation of viral genome replication MX1, OAS1, OAS3 3 3.70E-03 2.40E-01 GOTERM_MF_DIRECT double-stranded RNA binding OAS1, OAS2, OAS3 3 7.90E-03 2.30E-01 GOTERM_BP_DIRECT interferon-gamma-mediated signaling pathway OAS1, OAS2, OAS3 3 1.10E-02 4.20E-01 GOTERM_MF_DIRECT transferase activity OAS1, OAS2, OAS3 3 1.90E-02 3.70E-01 GOTERM_BP_DIRECT immune response OAS1, OAS2, OAS3 3 2.50E-01 1.00E+00 GOTERM_MF_DIRECT ATP binding MVK, NAV3, OAS1, OAS2, OAS3 5 4.20E-01 1.00E+00

Indacaterol-induced genes (defined by using Affymetrix probe sets) with significant expression level changes of <0.67-fold (based on a FDR P -value of <0.1) were subjected to functional annotation clustering using DAVID (default parameters). Clusters with enrichment scores >1.3 are shown.