Increased Cell Surface Fas Expression Is Necessary and Sufficient To Sensitize Lung Fibroblasts to Fas Ligation-Induced Apoptosis: Implications for Fibroblast This information is current as Accumulation in Idiopathic Pulmonary of October 1, 2021. Fibrosis Murry W. Wynes, Benjamin L. Edelman, Amanda G. Kostyk, Michael G. Edwards, Christopher Coldren, Steve D. Groshong, Gregory P. Cosgrove, Elizabeth F. Redente, Alison Bamberg, Kevin K. Brown, Nichole Reisdorph, Downloaded from Rebecca C. Keith, Stephen K. Frankel and David W. H. Riches J Immunol 2011; 187:527-537; Prepublished online 1 June 2011; doi: 10.4049/jimmunol.1100447 http://www.jimmunol.org/ http://www.jimmunol.org/content/187/1/527

Supplementary http://www.jimmunol.org/content/suppl/2011/06/01/jimmunol.110044 Material 7.DC1 References This article cites 62 articles, 18 of which you can access for free at: by guest on October 1, 2021 http://www.jimmunol.org/content/187/1/527.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Increased Cell Surface Fas Expression Is Necessary and Sufficient To Sensitize Lung Fibroblasts to Fas Ligation-Induced Apoptosis: Implications for Fibroblast Accumulation in Idiopathic Pulmonary Fibrosis

Murry W. Wynes,*,† Benjamin L. Edelman,* Amanda G. Kostyk,* Michael G. Edwards,† Christopher Coldren,† Steve D. Groshong,†,‡ Gregory P. Cosgrove,†,‡ Elizabeth F. Redente,* Alison Bamberg,*,x Kevin K. Brown,†,‡ Nichole Reisdorph,x Rebecca C. Keith,†,‡ Stephen K. Frankel,†,‡ and David W. H. Riches*,†,x

Idiopathic pulmonary fibrosis (IPF) is associated with the accumulation of collagen-secreting fibroblasts and myofibroblasts in the Downloaded from lung parenchyma. Many mechanisms contribute to their accumulation, including resistance to apoptosis. In previous work, we showed that exposure to the proinflammatory cytokines TNF-a and IFN-g reverses the resistance of lung fibroblasts to apoptosis. In this study, we investigate the underlying mechanisms. Based on an interrogation of the transcriptomes of unstimulated and TNF-a– and IFN-g–stimulated primary lung fibroblasts and the lung fibroblast cell line MRC5, we show that among Fas-signaling pathway molecules, Fas expression was increased ∼6-fold in an NF-kB– and p38mapk-dependent fashion. Prevention of the

increase in Fas expression using Fas small interfering RNAs blocked the ability of TNF-a and IFN-g to sensitize fibroblasts to http://www.jimmunol.org/ Fas ligation-induced apoptosis, whereas enforced adenovirus-mediated Fas overexpression was sufficient to overcome basal re- sistance to Fas-induced apoptosis. Examination of lung tissues from IPF patients revealed low to absent staining of Fas in fibroblastic cells of fibroblast foci. Collectively, these findings suggest that increased expression of Fas is necessary and sufficient to overcome the resistance of lung fibroblasts to Fas-induced apoptosis. Our findings also suggest that approaches aimed at increasing Fas expression by lung fibroblasts and myofibroblasts may be therapeutically relevant in IPF. The Journal of Immu- nology, 2011, 187: 527–537.

rogressive pulmonary fibrosis, especially idiopathic pul- unrestrained accumulation of fibroblasts and myofibroblasts that monary fibrosis (IPF), is thought to arise following injury synthesize and deposit collagen fibrils within fibroblast foci lo- by guest on October 1, 2021 P to, and abnormal repair of, the distal alveolar-capillary cated in the pulmonary parenchyma (2, 3). Recent studies have units (1). Although little is known about how the alveolar epi- suggested that pulmonary fibroblasts arise by several routes thelium is injured, the ensuing fibrotic response is associated with including increased migration and proliferation of resident pul- monary fibroblasts, mesenchymal transition of the alveolar epithelium, and recruitment of bone marrow-derived progenitor *Program in Cell Biology, Department of Pediatrics, National Jewish Health, Denver, cells (4–7). In the presence of TGF-b and other agonists, resident † CO 80206; Division of Pulmonary Sciences and Critical Care Medicine, Department fibroblast subsets transdifferentiate into a-smooth muscle actin- of Medicine, University of Colorado School of Medicine, Aurora, CO 80010; ‡Department of Medicine, National Jewish Health, Denver, CO 80206; and xDepart- positive myofibroblasts (8, 9), produce increased amounts of ment of Immunology, University of Colorado School of Medicine, Aurora, CO 80010 collagen, and, through their contractile activities, distort the pa- Received for publication February 11, 2011. Accepted for publication April 22, 2011. renchymal lung architecture (9, 10). Furthermore, in contrast to This work was supported by Public Health Service Grants HL068628 and HL055549 normal resolution of the repair process, in which fibroblasts and (to D.W.H.R.) and Grant T15 HL086386-01 (to N.R.) from the National Heart, Lung, myofibroblasts are eliminated by apoptosis (11), in situ studies and Blood Institute of the National Institutes of Health. M.W.W. was supported in part by a Parker B. Francis fellowship. A.B. was supported in part by T32 Training with fibrotic lung tissues from IPF patients and bleomycin-in- Grant AI07405 from the National Institute of Allergy and Infectious Diseases. E.F.R. duced pulmonary fibrosis in mice have shown that fibroblasts was supported by Ruth L. Kirschstein F32 National Research Service Award HL095274 from the National Heart, Lung, and Blood Institute and a Viola Vestal and myofibroblasts are resistant to apoptosis and accumulate in the Coulter scholarship from National Jewish Health. lung parenchyma (12–14). Remarkably, little is known about the The array data presented in this article have been submitted to the Gene Expression physiologic and pathologic mechanisms that contribute to fibro- Omnibus under accession number GSE26594. blast survival or to their susceptibility to apoptosis. Address correspondence and reprint requests to Dr. David W.H. Riches, Program in Fibroblast apoptosis can be induced by multiple agonists, cog- Cell Biology, Department of Pediatrics, Iris and Michael Smith Building Room nate receptors, and signaling pathways that converge to promote A549, National Jewish Health, 1400 Jackson Street, Denver, CO 80206. E-mail address: [email protected] caspase activation. Conversely, resistance to apoptosis occurs fol- The online version of this article contains supplemental material. lowing exposure of fibroblasts to an array of prosurvival factors Abbreviations used in this article: DISC, death-inducing signaling complex; FasL, including TGF-b (15, 16). Recent studies have shown that Fas, Fas ligand; FDR, false discovery rate; ILD, Interstitial Lung Disease; IPF, idiopathic a cell-surface death receptor of the TNFR superfamily, paradox- pulmonary fibrosis; MOI, multiplicity of infection; qPCR, quantitative RT-PCR; ically initiates both survival/differentiation and apoptosis in a siRNA, small interfering RNA. context-specific fashion. Ligation of Fas alone activates ERK and Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 NF-kB signaling pathways, promotes neuronal and epithelial cell www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100447 528 LUNG FIBROBLAST Fas EXPRESSION AND APOPTOSIS survival (17–19), and induces minimal apoptosis in lung fibro- the Advancement of Medicine (Edison, NJ), in accordance with an ap- blasts (20). In contrast, prior exposure to TNF-a and IFN-g ren- proved Institutional Review Board protocol (HS-1539). All donors suffered ders fibroblasts and myofibroblasts exquisitely sensitive to Fas brain death and were evaluated for organ donation before research consent. All lung samples failed regional lung selection criteria for transplantation. ligation-induced apoptosis and allows them to overcome the pro- Individuals had no evidence of current systemic or pulmonary infection, survival effect of TGF-b (20). Two recent studies have shown a clear chest radiograph, and partial pressure for oxygen/fraction of in- . a similar sensitizing effect of PGE2 on Fas-induced fibroblast spired oxygen ratio 250 mm Hg O2. Patients were excluded if they had apoptosis (14, 21). Although the source(s) of Fas ligand (FasL) are any history of lung disease or a history of systemic disease that commonly affects the lungs (e.g., rheumatoid arthritis or systemic lupus eryth- not completely known, recent studies have suggested that myofi- ematosus). Fibrotic lung fibroblasts from IPF patients were derived from broblasts themselves may be an important source in the fibrotic surgical lung biopsy specimens obtained as part of a prospective, In- lung (22, 23). Taken together, these findings suggest that fibroblast stitutional Review Board-approved, longitudinal study of the pathobiology and myofibroblast accumulation in the lungs of IPF patients could of fibrotic lung disease within the Interstitial Lung Disease (ILD) Program occur as a result of impaired sensitization to Fas-induced apo- at National Jewish Health (HS1603). Pathologic diagnoses were de- termined via review by an expert pulmonary pathologist with the ILD ptosis. They are also consistent with the developing notion that Program, and a pathologic diagnosis of usual interstitial pneumonia was fibrosis can progress in the absence of a robust inflammatory re- required for a clinical diagnosis of IPF. All clinical diagnoses were de- sponse associated with reduced levels of proinflammatory sensi- termined by an ILD Program physician who was caring for the patient, tizing molecules such as TNF-a, IFN-g, and PGE (1, 24–26). adhering to established pathologic and clinical criteria for the diagnosis of 2 idiopathic pulmonary fibrosis (27, 28). Freshly harvested explants, surgical Seeking to understand how TNF-a and IFN-g sensitize fibro- lung biopsies, or murine lungs were minced into 1 to 2 mm3 sections and blasts to Fas-induced apoptosis, the goal of this study was to cultured in DMEM containing 10% heat-inactivated FCS media on scored investigate the mechanisms that couple cytokine-induced sensiti- tissue-culture dishes. After 3 to 4 d, fibroblasts derived from the tissue zation to the acquisition of susceptibility to Fas-induced apoptosis. were trypsinized and maintained in tissue culture. All experiments were Downloaded from Based on an initial analysis of the transcriptomes of unstimulated performed on early passage (2–4) cell cultures. and TNF-a– and IFN-g–stimulated lung fibroblasts, we addressed RNA isolation, Northern, microarray, and quantitative PCR the functional necessity of increased Fas expression in cytokine- analysis induced sensitization of fibroblasts to Fas ligation-induced apo- Total RNA was isolated using TRIzol (Life Technologies, Grand Island, ptosis. In this study, we show that exposure to TNF-a and IFN-g NY). Northern blot analysis was performed as previously described (29). mapk http://www.jimmunol.org/ increases cell surface Fas expression in an NF-kB– and p38 - Prior to quantitative RT-PCR (qPCR) analysis, total RNA was cleaned up dependent fashion. Furthermore, through the use of small in- using the Qiagen RNeasy mini kit (Qiagen, Valencia, CA). Reverse tran- terfering RNA (siRNA)-mediated Fas knockdown and adenovirus- scription was performed on 1 mg total RNA with random hexamers in a 50- ml reaction using TaqMan RT reagents (Applied Biosystems). The single- mediated cell surface Fas expression, we show that among 603 plex qPCR reactions for Fas or GAPDH were performed using the probe/ genes for which expression was altered in response to TNF-a and primer sets Hs00531110_m1 and 4326317E, respectively, with 40 ng IFN-g, increased cell-surface expression of Fas is necessary and cDNA, 250 nM fluorogenic probe, and 900 nM each primer for Fas and sufficient to promote sensitization to Fas-induced apoptosis. Fur- 150 nM each primer for GAPDH. The specificity of PCR was verified by thermore, immunohistochemical staining of lung sections from the lack of signal in the no-template and no-reverse transcriptase controls. The threshold cycle was recorded for each sample. The relative Fas mRNA IPF patients revealed minimal expression of Fas by fibroblasts and expression levels were determined using the comparative threshold cycle by guest on October 1, 2021 myofibroblasts in fibroblastic foci. Collectively, these findings method, as previously described (30). Microarray analysis was conducted emphasize the importance of proinflammatory cytokines in the with 5 mg total RNA, which was labeled and hybridized to Affymetrix initiation of Fas-induced fibroblast apoptosis and suggest that the U133 Plus2.0 arrays (Affymetrix) using standard methods. The complete array data set and experimental details are available from the Gene Ex- underlying mechanism of their action involves raising Fas cell- pression Omnibus repository under accession number GSE26594 (http:// surface expression above a basal threshold. www.ncbi.nlm.nih.gov/geo/). Flow cytometry Materials and Methods Fas cell-surface expression was determined by flow cytometry. Lung fi- Materials broblasts were harvested by trypsinization and stained for 4 h on ice using Recombinant human and mouse TNF-a and IFN-g were purchased from either the APC-conjugated anti-Fas Ab or APC-conjugated nonimmune R&D Systems (Minneapolis, MN). Agonistic anti-human Fas (CH-11) and IgG. After washing, the cells were analyzed on an FACScalibur flow anti-mouse-Fas (Jo-2) Abs were from Upstate Biotechnology (Lake Placid, cytometer (BD Biosciences, San Jose, CA). For total Fas expression (cell NY) and BD Pharmingen (San Diego, CA), respectively. Fas probe and surface and intracellular), the cells were first fixed and permeabilized with primer sets (Hs00531110_m1) for quantitative real-time PCR were from 1.5% (w/v) paraformaldehyde and 0.1% (v/v) saponin, for 30 min, washed, Applied Biosystems (Foster City, CA). Human Genome U133 Plus 2.0 stained, and analyzed as above but with the inclusion of saponin in the microarray chips were purchased from Affymetrix (Foster City, CA). Fas stain and subsequent washes. The data were analyzed with FlowJo (Tree (sc715-R) and IkB-a (sc371R) Western Abs were from Santa Cruz Bio- Star, Ashland, OR) and Cell Quest software (BD Biosciences). technology (Santa Cruz, CA), the actin (mab1501) Western Ab was from Western blotting Millipore (Temecula, CA) and the phospho-specific HSP27 (PA1-005) Ab was from Affinity Bioreagents (Golden, CO). Conjugated Abs for flow Protein expression in cell lysates was determined by Western blot analysis. cytometry (allophycocyanin-labeled Fas Ab [558814] and control Ab Four hundred microliters lysis buffer (50 mM Tris-HCl [pH 7.4] containing mapk [555751]) were from BD Pharmingen. The inhibitors SB 203580 (p38 ) 150 mM NaCl, 1 mM Na2EDTA, 1% [v/v] Nonidet P-40, 0.1% [w/v] and Bay 11-7082 (NF-kB, PD98059 [MEK1] and LY294002 [PI3K]) were SDS, 1% [w/v] sodium deoxycholate, 20 mM b-glycerophosphate, 1 purchased from Calbiochem (San Diego, CA). mM Na3VO4,1mg/ml leupeptin, 2 mg/ml aprotinin, and 1 mM PMSF) were added to each well of a six-well plate. The cells were scraped off the Cell culture plate, and 10 mg protein from the postnuclear lysate was analyzed as Human primary lung fibroblasts from an IPF patient (FS087) and non- previously described (31). disease control subject (N78), the human fetal lung fibroblast cell lung Fas knockdown and adenovirus-mediated expression MRC-5 (American Tissue Culture Collection, Manassas, VA), and mouse primary lung fibroblasts were maintained in modified Eagle’s MEM, Fas knockdown was achieved in MRC5 cells using a pool of four Fas- supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% specific Accell SMARTpool siRNAs (E-003776-00-0010 human Fas, (v/v) heat-inactivated FBS under a 5% CO2 atmosphere as previously NM_152876; Dharmacon). Fas siRNAs or a nontargeting Accell control described (20). Primary cultures of human lung fibroblasts were derived siRNA (D-001910-10-20) were prepared according to the manufacturer’s from nondiseased human lungs obtained from the International Institute for instructions (Dharmacon) and incubated with MRC5 cells in six-well The Journal of Immunology 529 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Genome-wide mRNA expression responses to TNF-a and IFN-g stimulation in three human lung fibroblast cell lines. Two primary (FS087 and N78) and one immortalized (MRC5) cell line were stimulated with TNF-a and IFN-g, and transcriptional responses were measured using Affymetrix Human Genome U133 Plus 2.0 GeneChips. A, Principal components analysis plot representing global gene expression profile of control (red) and cytokine- stimulated (blue) fibroblast cell lines. B, Venn diagram showing commonality of transcripts with 2-fold or greater changes in expression between the stimulated and unstimulated states for the three different fibroblast cell lines. C, Distribution of p values generated from an unpaired t test of stimulated versus control cells. D, Immunological and apoptotic pathways are prevalent in genes differentially expressed with cytokine stimulation among the 10 most widely overrepresented pathways associated with the list of genes differentially expressed (FDR ,0.05) upon cytokine stimulation. The negative log of the p values for the probability of obtaining these genes associated with the given pathways by random chance is given at the top, whereas the bar line in the bottom graph is derived from the ratio of the number of genes on our list associated with a given pathway divided by the total number of genes that make up that pathway. The threshold line corresponds to a p value of 0.05. E, Detailed view of the canonical pathway death receptor signaling containing genes upregulated with cytokine stimulation. Genes colored red/pink indicate significant increases in expression (FDR ,0.05) upon cytokine stimulation. Ex- pression fold increases are indicated. A total of six arrays were analyzed. plates in delivery medium at a final concentration of 1 mM. Additional a further 36 h. The cells were then either stained for cell-surface Fas ex- plates of control cells were grown in parallel. After 36 h, TNF-a (10 ng/ pression or incubated with agonistic anti-Fas Ab (CH-11; 250 ng/ml) for ml) and IFN-g (50 U/ml) were added, and the incubations continued for 4 h and assessed for caspase-8 activation, as described above. 530 LUNG FIBROBLAST Fas EXPRESSION AND APOPTOSIS

Enforced cell-surface expression of Fas was accomplished using an ml Eagle’s modified MEM containing 10% (v/v) heat-inactivated FCS were adenovirus expressing human Fas under the control of the chicken actin added, and 24 h later, the cells were placed in medium containing 0.1% (v/v) promoter. The adenoviral construct was created by ligating EcoRI and ClaI- heat-inactivated FCS and incubated for a further 36 h prior to analysis. digested full-length human Fas into EcoRI and ClaI pAd5 CMV GFP vector to create a bistronic construct in which Fas and GFP were separated by an Immunohistochemistry internal ribosome entry site. Next, the majority of the CMV enhancer was excised by digestion with NdeI and ClaI, and NdeI/ClaI-digested chicken Lung tissues were fixed with 10% (v/v) formalin and embedded in paraffin. b-actin promoter was ligated in place of the CMV promoter to yield the Each block was sequentially cut into 4-mm-thick serial sections on final construct in which Fas-GFP is transcribed under control of the Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA) and prepared for chicken b-actin promoter. The fidelity of the construct was confirmed by: immunohistochemical staining as previously described (13). Sections were 1) diagnostic restriction enzyme digestion; 2) nucleotide sequencing; and stained with: 1) anti-Fas (Santa Cruz Biotechnology; Ab SC 715) used at 3) expression of Fas and GPF in HEK293 cells as determined by Western a 1:100 dilution; or 2) anti-human a-smooth muscle actin Ab (Sigma- blotting and microscopy to visualize GFP-labeled cells. Adenoviruses Aldrich, St. Louis, MO; A2547) used at a 1:200 dilution. Negative con- expressing the Fas-GFP bistronic sequence were prepared by the Gene trols consisted of nonimmune mouse or rabbit IgG and were used at Transfer Vector Core at the University of Iowa (http://www.uiowa.edu/ the same concentration as the primary Abs. Immunohistochemistry was ∼gene/). MRC cells plated in six-well plates were transduced with Fas and performed either manually or using an automated immunohistochemical empty vector adenoviruses suspended in 1 ml Optimem (Invitrogen) for 1 h stainer (DakoCytomation, Carpinteria, CA) in which slides were incubated at 37˚C. Preliminary experiments were conducted to determine the level sequentially with each primary or control Ab (30 min, 25˚C) followed by of cell surface Fas expression achieved by transduction with the Fas- secondary Ab conjugated to polymerized HRP enzymatic domains (DAKO adenovirus at multiplicities of infection (MOI) of 10, 30, 100, and 300. Envision FLEX system; DakoCytomation), and chromogenic enzyme sub- Fas expression was found to be slightly higher than the level seen fol- strate (4 min, 25˚C) according to the manufacturer’s protocol. The slides lowing coincubation with TNF-a (10 ng/ml) and IFN-g (50 U/ml) fol- were counterstained with hematoxylin (4 min, 25˚C), rinsed in water, lowing transduction with the Fas adenovirus at an MOI of 300, and so this dehydrated with graded alcohols/xylene, and coverslipped with permanent MOI was used in all experiments. Following transduction, an additional 2 mounting solution. Downloaded from http://www.jimmunol.org/

FIGURE 2. Stimulation with TNF-a and IFN-g in- creases Fas mRNA, protein, and cell-surface expres- sion in human fibroblasts. A, Exposure of MRC-5 cells to TNF-a 10 ng/ml) plus IFN-g (50 U/ml) increases Fas mRNA abundance as determined by qPCR (left panel) and Northern analysis (center pa- nel). The right panel shows quantification of Fas mRNA levels as determined by densitometry of

Northern blots. B, Increased expression of total Fas by guest on October 1, 2021 protein by MRC-5 cells exposed to TNF-a 10 ng/ml) plus IFN-g (50 U/ml). Upper panel shows Western blots probed for Fas and b-actin; lower panels show the fold increase in total Fas protein compared with unstimulated cells. C, TNF-a 10 ng/ml) plus IFN-g (50 U/ml) induced increased to cell-surface Fas in MRC-5 cells. Upper panel shows a representative flow cytometry histogram illustrating the patterns of Fas staining. Lower panels show the increase (fold over unstimulated) in cell-surface Fas expression. D, Fas is not stored in an intracellular pool prior to translocation to the cell surface. Left panel, Repre- sentative flow cytometry histograms of MRC5 cells cultured in medium alone and then stained with an allophycocyanin-labeled Fas-specific Ab under conditions that maintain plasma membrane integrity (surface) or after permeabilization and fixation (to- tal). Center panel, Representative flow cytometry histograms of unstimulated and TNF-a (10 ng/ml) plus IFN-g (50 U/ml)-stimulated MRC-5 cells stained with APC-labeled anti-Fas Ab under con- ditions that maintain cell integrity (surface) or after permeabilization and fixation (total). Right panel, Quantification of surface and total Fas staining in unstimulated and TNF-a plus IFN-g–stimulated MRC-5 cells. Data represent the mean 6 SEM of three independent experiments. The Journal of Immunology 531

Statistical analysis (10 ng/ml) and IFN-g (50 U/ml) for up to 48 h and measured All experiments were conducted a minimum of three times. Statistical Fas mRNA levels by qPCR and Northern analysis. qPCR analysis analyses using GraphPad InStat version 3.0b for Macintosh (GraphPad, San showed that Fas mRNA expression peaked at ∼6-fold above basal Diego CA) were performed by one-way ANOVA, and comparisons among levels by 24 h and declined to ∼4-fold above basal levels at 48 h groups were performed with a Newman-Keul multiple comparison test. A p (Fig. 2A). Similarly, Northern analysis indicated that Fas mRNA , value 0.05 was considered to be significant. All microarray data met the was increased ∼3.5 fold following 36 h of stimulation with TNF-a quality control criteria established by the Tumor Analysis Best Practices Working Group (32). Microarray data were analyzed in Partek Genomics and IFN-g (Fig. 2A). Suite (Partek, St. Louis, MO), using GC robust multiarray average probe set summarization. Transcripts that were differentially expressed between cytokine-stimulated and control fibroblasts were identified using a Student t test, with a significance threshold defined by the 5% false discovery rate (FDR). Biological themes differentially affected by cytokine stimulation were identified among the 603 differentially expressed transcripts using a modified Fischer’s exact test as implemented in Ingenuity Pathway Analysis (http://www.ingenuity.com).

Results Changes in normal and IPF lung fibroblast transcriptomes induced by TNF-a and IFN-g To investigate the mechanism by which TNF-a and IFN-g re- Downloaded from program fibroblasts from resistance to sensitivity to Fas ligation- induced apoptosis, we exposed representative normal and fibrotic human lung fibroblasts and the human lung fibroblast cell line MRC5 to the combination of TNF-a (10 ng/ml) and IFN-g (50 U/ ml) for 36 h and analyzed changes in their transcriptomes using Affymetrix microarrays. We observed that the transcriptional re- sponses to cytokine stimulation were large and consistent within http://www.jimmunol.org/ the set of fibroblastic cells examined. Approximately 50% of the transcripts with 2-fold or greater expression change upon stimu- lation within a single fibroblast line were likewise changed in all three fibroblast groups (Fig. 1B). This consistent pattern of gene expression change is visible in an unsupervised principal com- ponents analysis (Fig. 1A). In this plot, each experimental sample is represented as a solid shape for which the position in space

is determined by genome-wide transcript levels. The consistent by guest on October 1, 2021 pattern of expression changes upon stimulation appears as a vector of similar orientation and length connecting the control and stim- ulated samples from each line. The most robustly induced family of transcripts was chemokine receptor ligands (e.g., CCL20, CCL5, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL3, and CXCL9), which exhibited nominal increases from 18.2- to 5930- fold. A statistically defined set of 603 differentially expressed tran- scripts was identified using a Student t test, with a significance threshold determined by the 5% FDR (Supplemental Table I). Biological themes underlying this expression pattern were iden- tified based on the overrepresentation of predefined groups of transcripts within the statistically defined set. As expected, the most overrepresented canonical pathways (Fig. 1D) include pathways associated with IFN signaling, the inflammatory re- FIGURE 3. Increased Fas expression and sensitization to Fas ligation- sponse, and death receptor signaling (Fig. 1E). Among the latter, induced apoptosis by TNF-a and IFN-g is dependent on signaling by TNF- Trail (APO2L) mRNA expression was increased 1051-fold, R1, p38mapk, and NF-kB. A, TNF-a and IFN-g induce Fas expression whereas expression of the antiapoptotic genes cIAP and c-FLIP by primary mouse lung fibroblasts from wild-type mice and TNF-R22/2 2 2 was increased 132-fold and 9.1-fold, respectively. Notably, within mice, but not TNF-R1 / mice. B, TNF-a and IFN-g sensitize primary 2 2 the Fas signaling pathway, Fas expression was increased 6.1-fold, mouse lung fibroblasts from wild-type and TNF-R2 / mice, but not TNF- 2/2 mapk whereas transcripts encoding other members of the Fas-signaling R1 mice, to Fas ligation-induced apoptosis. C, The p38 inhibitor cascade were not affected by cytokine stimulation. Together, these SB203580 (20 mM) blocks TNF-a and IFN-g induction of Fas expression in human MRC-5 lung fibroblastic cells. D, Inhibition of TNF-a and IFN-g findings suggest that the level of Fas expression might play induce Fas expression by MRC fibroblastic cells in the presence of the NF- a dominant role in the sensitization to Fas ligation-induced apo- kB inhibitor Bay 11-7082 (3 mM). E, The p38mapk inhibitor SB203580 (20 ptosis. mM) inhibits TNF-a and IFN-g sensitization of MRC-5 lung fibroblastic Fas expression is increased in response to TNF-a and IFN-g cells to Fas ligation-induced apoptosis. F, TNF-a and IFN-g sensitization to Fas-induced apoptosis is inhibited by the NF-kB inhibitor Bay 11-7082 Based on the findings from the microarray experiments, we con- (3 mM). The data shown in the upper panels of C and D are representative firmed the effect of TNF-a and IFN-g on Fas mRNA levels by data from a single experiment. The lower panels show the mean 6 SEM stimulating human lung fibroblastic MRC5 cells with TNF-a from three independent experiments. 532 LUNG FIBROBLAST Fas EXPRESSION AND APOPTOSIS

We next measured the effect of TNF-a and IFN-g on expression cellular Fas staining was the same as cell-surface staining in of Fas protein. MRC5 cells were incubated in either medium alone unstimulated cells. In addition, the TNF-a– and IFN-g–induced or with TNF-a (10 ng/ml) and IFN-g (50 U/ml) for up to 36 h. The increase in total Fas and cell-surface Fas staining was identical cells were then lysed and evaluated for Fas expression by Western (Fig. 2D, center and right panels), suggesting that Fas is expressed blotting (Fig. 2B) or harvested and subjected to flow cytometry at the cell surface and not associated with an intracellular pool. analysis following staining with allophycocyanin-conjugated anti- Together, these findings indicate that TNF-a and IFN-g increase Fas Ab (Fig. 2C). Both approaches indicated that stimulation with Fas transcription and translation and lead to increased cell-surface TNF-a and IFN-g for 36 h resulted in an ∼6-fold increase in total expression of Fas.

Fas protein levels and cell-surface Fas expression. In addition, Fig. mapk 2C (upper panel) shows that the increase in Fas expression in Signaling via TNF-R1, p38 and NF-kB are required for response to TNF-a and IFN-g was seen in a single population of increased Fas expression and sensitization to Fas cells. ligation-induced apoptosis Previous studies have suggested that Fas is localized both at the TNF-a–initiated signaling involves two receptors (TNF-R1, TNF- cell surface and within intracellular organelles, including the Golgi R2) and multiple signaling pathways (35). To investigate the role complex (33, 34). Although the data shown in Fig. 2A–C suggest of TNF-R1 and TNF-R2 in TNF-a– and IFN-g–induced fibroblast that the increase in cell-surface Fas expression is most likely due sensitization to Fas-induced apoptosis, primary cultures of lung 2 2 2 2 to a coordinated increase in Fas transcription, translation, and fibroblasts from TNF-R1 / , TNF-R2 / , and C57BL/6 mice translocation to the cell surface, they do not completely rule out were stimulated with TNF-a (10 ng/ml), IFN-g (50 U/ml), or both the possibility that translocation of a pre-existing intracellular cytokines for 48 h and analyzed for cell-surface Fas expression by pool contributes to the TNF-a– and IFN-g–dependent increase in flow cytometry. Fig. 3A shows that stimulation with TNF-a alone Downloaded from cell-surface Fas. To address this possibility, we compared total and TNF-a plus IFN-g significantly increased Fas expression on 2 2 cellular Fas levels (intracellular and cell-surface) and cell-surface fibroblasts from TNF-R2 / mice (p , 0.01) and C57BL/6 mice Fas levels by flow cytometry in detergent-permeabilized and non- (p , 0.01), but failed to increase in Fas expression on lung 2 2 permeabilized cells. Fig. 2D (left and right panels) shows that total fibroblasts from TNF-R1 / mice (p . 0.05). Similarly, Fig. 3B http://www.jimmunol.org/

FIGURE 4. The TNF-a– and IFN-g–induced in- by guest on October 1, 2021 crease in Fas expression is necessary to sensitize lung fibroblastic cells to Fas ligation-induced apoptosis. A, Fas siRNA inhibits the TNF-a plus IFN-g–induced increase in Fas expression in human MRC-5 fibro- blastic cells without affecting basal Fas expression. The cells were incubated with Fas-specific or control siRNAs (1 mM) for 36 h prior to stimulation with TNF-a (10 ng/ml) plus IFN-g (50 U/ml) for 36 h. The left panel shows a representative flow cytometry his- togram. The right panel shows the fold changes in Fas expression. B, Prevention of the TNF-a plus IFN-g–induced increase in cell-surface Fas expression using a Fas-specific siRNA inhibits sensitization to Fas ligation-dependent apoptosis. Upper panels show representative flow cytometry histograms for active caspase-8 in cells treated with cytokines and Fas li- gation in the absence of siRNAs (left panels) or in the presence of a control siRNA (right panels). The lower left panel shows representative flow histograms for cells treated with Fas siRNA prior to and during ex- posure to cytokines 6 Fas ligation. The lower right panel shows the quantification of apoptosis in cells treated with no siRNA, control siRNA, and Fas siRNA. The bars represent the means 6 SEM of three experiments. The Journal of Immunology 533 shows that lung fibroblasts from TNF-R12/2 mice were not sen- though incompletely, inhibited cytokine-induced sensitization to sitized to Fas-induced apoptosis (p . 0.05). Interestingly, com- Fas-induced apoptosis (p , 0.01 and p , 0.01, respectively; Fig. pared with fibroblasts from wild-type C57BL/6 mice, lung fi- 3E,3F). In control experiments, the concentrations of SB203580 broblasts from TNF-R22/2 mice exhibited a significant increase in and Bay11-7082 were found to completely block p38mapk and caspase-8 activation following stimulation with TNF-a alone (p , NF-kB activation as reflected by inhibition of the phosphorylation 0.05) and trended toward a significant increase in response to of IkBa and Hsp-27, respectively (Supplemental Fig. 1). Collec- TNF-a plus IFN-g (p = 0.08). Together, these data indicate that tively, these findings suggest that the TNF-a– and IFN-g–induced signaling via TNF-R1 is required for cytokine-induced upregula- increases in Fas expression and sensitization to Fas ligation- tion of Fas and sensitization to Fas-induced apoptosis. In addition, induced apoptosis require occupation of TNF-R1 and signaling signaling via TNF-R2, although not being required for Fas ex- via p38mapk and NF-kB. pression, delivers an antiapoptotic signal that reduces the level of apoptosis induced by ligation of TNF-R1. Increased Fas expression is necessary and sufficient to sensitize Next, we investigated the role of NF-kB, Akt, ERK, and p38mapk fibroblasts to Fas-induced apoptosis signaling in the TNF-a– and IFN-g–induced increase in Fas ex- To determine if the increase in Fas expression was necessary for pression and Fas-induced apoptosis using pharmacologic inhib- cytokine-induced sensitization to Fas-induced apoptosis, we used itors. Incubation of MRC5 cells with TNF-a and IFN-g in the siRNA knockdown to block the TNF-a and IFN-g increase in presence of the PI3K and MEK1 inhibitors LY294002 (20 mM) cell-surface Fas expression. We then determined the effect of and PD98059 (20 mM) failed to block the cytokine-induced in- Fas knockdown on cell-surface Fas expression and Fas ligation- crease Fas expression (data not shown). In contrast, pharmaco- induced caspase-8 activation. MRC5 cells were incubated with logic inhibition of p38mapk and NF-kB with SB203580 (20 mM, Fas or control siRNAs (1 mM) or medium alone for 36 h. Next, Downloaded from Fig. 3C) and Bay 11-7082 (3 mM, Fig. 3D), respectively, signifi- TNF-a (10 ng/ml) and IFN-g (50 U/ml), or medium alone, were cantly reduced the TNF-a– and IFN-g–induced increase in Fas added to the cells and the incubations continued for an additional expression (SB203580, p , 0.001; Bay11-7082, p , 0.001), 36 h. The cells were then evaluated for cell-surface Fas expres- suggesting that both p38mapk and NF-kB are required for the sion. Fig. 4A shows that neither Fas siRNA nor control siRNA TNF-a– and IFN-g–induced increase in Fas expression. Similarly, significantly affected basal cell-surface Fas expression (p . 0.05 pharmacologic inhibition of p38mapk and NF-kB significantly, and p . 0.05, respectively). However, whereas the control siRNA http://www.jimmunol.org/

FIGURE 5. Increased cell-surface Fas expression

is necessary to sensitize human lung MRC-5 fibro- by guest on October 1, 2021 blastic cells to Fas ligation-induced apoptosis. A, Representative flow histogram illustrating increased cell-surface Fas expression following transduction of human MRC-5 fibroblastic cells with an adenovirus encoding human Fas. The induction of Fas by TNF-a and IFN-g is also shown as a control. B, Represen- tative flow cytometry histogram showing that liga- tion of adenovirus Fas-transduced MRC-5 cells was sufficient to permit Fas ligation-induced apoptosis, though less efficiently than TNF-a plus IFN-g. Quantification of Fas ligation-induced apoptosis (measured by Annexin V staining) in adenovirus Fas and empty adenovirus-transduced MRC-5 cells in the absence of TNF-a plus IFN-g. Sensitization to Fas-induced apoptosis by TNF-a plus IFN-g is shown as a comparison. C, A graph illustrating the relationship between increasing quartiles of adeno- virus-mediated Fas expression (0–50 [1st quartile], 50–100 [2nd quartile], 100–150 [3rd quartile], and 150–200 [4th quartile]) and Fas ligation-induced apoptosis suggests that the ability to undergo Fas- induced apoptosis is directly correlated with the level of cell-surface Fas expression. The data represent the mean 6 SEM of three independent experiments. *p . 0.05 between the 1st and 2nd quartiles, **p , 0.05 between the 1st and 3rd quartiles, ***p , 0.01 be- tween the 1st and 4th quartiles. 534 LUNG FIBROBLAST Fas EXPRESSION AND APOPTOSIS had no effect on cytokine-induced Fas expression (p . 0.05), overcomes the basal resistance of lung fibroblasts to Fas ligation- incubation with Fas siRNA almost completely blocked the in- induced apoptosis, we next investigated Fas expression in the fi- crease seen in response to TNF-a and IFN-g (p , 0.01) (Fig. 4A). broblastic foci of lung tissues from IPF patients by immunostaining Next, we investigated the effect of Fas knockdown on Fas ligation- paraffin sections of lung tissues from IPF patients with an anti-Fas induced caspase-8 activation. Fig. 4B shows that compared with Ab. As shown in Fig. 6A,6C, and 6D, we observed low to minimal cells incubated in the absence of siRNAs or incubated with control Fas staining by fibroblasts and myofibroblasts in the fibroblastic siRNA, specific Fas knockdown resulted in a complete inhibi- foci of IPF patients, with only occasional positive Fas staining by tion of Fas-induced caspase-8 activation in TNF-a– and IFN-g– interstitial macrophages. In contrast, Fas was abundantly detected exposed cells. These data indicate that the cytokine-induced in- in type I and type II cells overlying the fibroblastic foci, as well as crease in cell-surface Fas expression is required for Fas ligation- in alveolar macrophages (Fig. 6A,6C) and lymphocytes clustered induced caspase-8 activation and apoptosis in fibroblasts. in lymphoid aggregates (Fig. 6A). Fas was detected primarily in To determine if increased Fas expression was sufficient to alveolar macrophages in the lung parenchyma of nondiseased sensitize fibroblasts to Fas ligation-induced apoptosis, we con- control subjects (Fig. 6E). structed an adenovirus expressing human Fas under the control of the chicken b-actin promoter and transduced MRC5 cells with Discussion Fas-expressing or empty vector adenoviruses at an MOI of 300 Building on earlier work showing that the proinflammatory Th1 for 36 h. Fig. 5A shows that transduction with the Fas adenovirus cytokines TNF-a and IFN-g reprogram lung fibroblasts and myo- led to an ∼8-fold increase in cell-surface Fas expression, where- fibroblasts from resistance to sensitivity to Fas-induced apoptosis, as transduction with the empty vector adenovirus had no effect the goal of the current study was to investigate the mechanisms Downloaded from on Fas expression. We next determined the effect of adenovirus- underlying this phenotypic change. In this study, we show for the mediated increased Fas expression on Fas ligation-induced ap- first time, to our knowledge, that among 603 transcripts for which optosis. In these experiments, apoptosis was quantified by mea- expression was reprogrammed by TNF-a and IFN-g, increased suring exposure of phosphatidylserine using allophycocyanin- conjugated Annexin V because the intrinsic fluorescence of adenovirus-encoded GFP was found to interfere with the caspase- 8 activation assay. Fig. 5B shows that following transduction with http://www.jimmunol.org/ the Fas adenovirus, MRC5 cells exhibited increased Fas ligation- induced Annexin V binding compared with cells transduced with empty vector adenovirus (p , 0.01). Of interest, we noticed that whereas the level of cell-surface Fas expression achieved by transduction with Fas-expressing adenovirus was greater than that induced by TNF-a and IFN-g (Fig. 5A), the level of Fas ligation- induced apoptosis was lower in cells transduced with the Fas adenovirus compared with fibroblasts exposed to TNF-a and IFN- by guest on October 1, 2021 g (Fig. 5B). These findings suggest that although increased cell- surface Fas expression is sufficient to render the cells susceptible to Fas-induced apoptosis, additional mechanisms induced by TNF-a and IFN-g may contribute to the efficiency of the sensi- tization. To explore this notion further, we investigated the re- lationship between the level of adenoviral-transduced cell-surface Fas expression and sensitivity to Fas ligation-induced apoptosis by quantifying the sensitivity to Fas ligation-induced apoptosis at incremental quartiles of Fas expression. Fig. 5C shows that the induction of apoptosis following Fas ligation increased linearly with increasing cell-surface Fas expression, and the slopes of the lines obtained in the presence and absence of Fas ligation were significantly different (p , 0.001). However, significant increases in Fas ligation-induced apoptosis were only detected in the third and fourth quartiles of Fas expression (p , 0.05 and p , 0.01, respectively). Taken together, these data suggest that Fas expres- sion must exceed a threshold level before the cells can undergo Fas ligation-induced apoptosis. In addition, because the level of FIGURE 6. Minimal Fas expression by the fibroblastic cells of fibroblast Fas expression seen following transduction with the Fas adeno- foci in IPF. A–D show lung tissues from patients with IPF, whereas E and F virus was higher than that induced by cytokine stimulation, the are from nondiseased human control subjects. A, C, D, and E were data provide further support to the notion that exposure to TNF-a immunohistochemically stained for Fas, whereas B was stained for a- and IFN-g also reduces the threshold for Fas ligation-induced smooth muscle actin to aid in the localization of Fas in the serial section apoptosis. shown in A. F illustrates lung tissue from a human nondisease control subject stained with nonimmune IgG as a negative control. Fas was Fas is minimally expressed by fibroblastic cells in lung tissues detected in alveolar epithelial cells (A, C), alveolar macrophages (A, D), from IPF patients and lymphocytes forming lymphoid aggregates (C). Fas was minimally detected or absent from the fibroblastic cells of fibroblast foci (A, C, and at Our overall rationale for investigating the mechanisms of sensi- higher magnification in D). Fas was only detected in alveolar macrophages tization of fibroblasts to Fas ligation-induced apoptosis was to learn in lung tissues to human nondisease control subjects (E). The images more about how this process is impaired in the fibroblastic foci of shown are representative Fas staining from 20 IPF patients (original IPF patients. Given our data showing that increased Fas expression magnification 3200). The Journal of Immunology 535 cell-surface expression of Fas was necessary and sufficient to other cell types have suggested that activation of NF-kB, c-Jun, overcome the basal resistance of lung fibroblastic cells to Fas and p53 (36–38) contribute to increased Fas expression. In addi- ligation-induced apoptosis. In addition, we show that the tion, p38mapk has been implicated in arachidonic acid-induced Fas apoptosis-resistant phenotype of fibroblastic cells in the fibroblast expression in U937 cells (39). However, although activation of foci of IPF/usual interstitial pneumonia patients is associated with NF-kB and p38mapk are key steps in increased Fas expression and low Fas expression. Together, these findings suggest that thera- sensitization to apoptosis induced by TNF-a and IFN-g, additional peutic interventions aimed at increasing Fas expression by fibrotic signaling mechanisms are likely to be involved because IL-1b and lung fibroblasts may reverse their state of basal resistance and may IL-6 both activate NF-kB and p38mapk yet fail to induce sensiti- potentially provide a new avenue to reduce fibroblast accumula- zation to Fas ligation-dependent apoptosis (S.K. Frankel and tion in the lungs of IPF patients. D.W.H. Riches, unpublished observations). Interestingly, although The dramatic and durable phenotypic changes in fibroblasts our studies with TNFR-deficient lung fibroblasts revealed that li- exposed to TNF-a and IFN-g are reflected in the large number of gation of TNF-R1 was required for the increase in Fas expression transcriptional changes this exposure elicits. These expression and susceptibility to Fas ligation-induced apoptosis, they also changes are similar in direction and magnitude in each of the three suggest a role for TNF-R2 in protecting the cells from apoptosis as cell lines for a majority of transcripts, even though the cell lines previously reported (40). Furthermore, because ligation of TNF- originate from vastly different lungs, suggesting that reprogram- R2 has also been shown to activate NF-kB and p38mapk signaling ming by TNF-a and IFN-g is a fundamental pathway open to lung (41), it is plausible that the incomplete inhibition of caspase-8 fibroblasts in multiple contexts. The biological themes that these activation seen in the presence of Bay11-7082 and SB203580 expression changes represent include those that are likely to be the may be related to loss of antiapoptotic signaling initiated by li- direct result of cytokine stimulation (i.e., the activation of IFN gation of TNF-R2. Collectively, our findings suggest that although Downloaded from signaling and inflammatory responses). In contrast, changes as- p38mapk and NF-kB activation are necessary in TNF-a– and IFN- sociated with genes involved in death receptor signaling appear to g–induced Fas expression and sensitization to apoptosis, their reflect a change in the poise of the cell with respect to signaling activation is unlikely to be sufficient to induce these responses. by these receptors rather than activation of this pathway. In- Thus, additional, and as yet unknown, mechanisms likely con- terestingly, expression of transcripts encoding the antiapoptotic tribute to the control of fibroblast apoptosis. molecules cIAP and c-FLIP was found to increase in response to Based on in vivo studies in mice (42, 43), we used an siRNA http://www.jimmunol.org/ TNF-a and IFN-g and may afford protection against the induction approach to show that increased Fas expression was necessary for of apoptosis by cytokines during the sensitization process. How- TNF-a– and IFN-g–induced sensitization to Fas ligation-induced ever, their expression is clearly not sufficient to protect the cells apoptosis. In addition, we showed that adenovirus-mediated over- from apoptosis following Fas ligation. Taken together, the expression of Fas was sufficient to render fibroblasts sensitive to microarray data suggest that fibroblast programming by TNF-a Fas ligation-induced apoptosis. However, although the level of Fas and IFN-g does not result in autocrine activation of death receptor expression achieved following transduction with the Fas- signaling, but reflects changes that may underlie their increased expressing adenovirus was higher than that induced by TNF-a sensitivity to Fas ligation. and IFN-g, the level of apoptosis detected in cells expressing the by guest on October 1, 2021 Among the genes for which expression was increased by TNF-a Fas adenovirus was lower than that induced by cytokines. These and IFN-g, Fas mRNA and cell-surface protein expression in- findings suggest that although increased Fas expression is neces- creased ∼5- to 6-fold. Both the increase in Fas expression and sary and sufficient to enable fibroblasts to undergo Fas ligation- sensitivity to Fas ligation-induced apoptosis were found to be induced apoptosis, other genes for which expression are differ- dependent on ligation of the TNFR TNF-R1 and activation of entially regulated by TNF-a and IFN-g may also contribute to p38mapk and NF-kB. Although little is known about transcriptional reprogramming fibroblast sensitivity to Fas ligation. In particular, regulation of Fas expression in lung fibroblasts, previous studies in as has been reported for several receptors including T cell and

FIGURE 7. Schematic illustrating the proposed mechanisms of sensitization of lung fibroblasts to apoptosis. A, Proposed mechanism controlling TNF-a– and IFN-g–induced Fas expression by lung fibroblasts. TNF-R1 ligation is proposed to induce sensitization to Fas ligation-induced apoptosis in two steps. Step 1 involves dissociation and/or reduced expression of a Fas-associated inhibitor of DISC assembly. Step 2 involves TNF-R1–induced increased cell- surface Fas expression via p38mapk and NF-kB signaling. Step 2 is bypassed by adenovirus-induced Fas expression, whereas step 1 is not. Step 1 is proposed to control threshold sensitivity to Fas-induced apoptosis. B, Proposed mechanisms underlying the persistence (resistance) and sensitivity of lung fibroblasts to Fas ligation-induced apoptosis. Basal (low) fibroblast Fas expression fails to induce Fas ligation-induced apoptosis (upper left panel), and fibroblastic cells persist in the fibroblastic foci of IPF patients (upper right panel). TNF-a and IFN-g increase Fas expression and enable Fas-induced apoptosis (lower left panel), thereby promoting a reduction in the burden of fibroblastic cells in fibroblastic foci (lower right panel). Left panels, Blue rectangles, Fas; red squares, soluble or cell-surface FasL. 536 LUNG FIBROBLAST Fas EXPRESSION AND APOPTOSIS

B cell Ag receptors (44, 45), our findings suggest that until Fas growth, differentiation, and chemokine production (57–62). In expression exceeds a certain threshold, its ability to signal apo- contrast, when Fas is expressed at comparatively high levels (e.g., ptosis remains blocked. As illustrated in Fig. 7A, an intriguing in hepatocytes and TNF-a– and IFN-g–stimulated lung fibro- possibility is that one of these putative additional gene(s) may blasts) (20, 42), its ligation results in DISC assembly and in- contribute to the control of this threshold by preventing death- duction of apoptosis. Thus, as illustrated schematically in Fig. 7B, inducing signaling complex (DISC) assembly at low levels of we speculate that resistance or sensitivity of lung fibroblasts to Fas Fas expression. ligation-induced apoptosis is controlled by the level of cell-surface Because apoptotic fibroblastic cells are rarely seen in the fi- Fas expression, even if FasL is ubiquitously present. It is also broblast foci of IPF patients (12, 13), and lung fibroblasts are possible that expression of FasL on the surface of myofibroblasts largely resistant to Fas-induced apoptosis when isolated and may allow the cells to interact with Fas on adjacent fibroblasts and studied in vitro (14, 20, 46), we investigated the level of Fas ex- other cell types in trans, suggesting that cell–cell contact and the pression in lung tissue from patients with IPF. Consistent with three-dimensional structure of the fibroblastic foci may play an previous reports, Fas was strongly expressed by alveolar epithelial important role in the regulation of Fas signaling and function. cells and alveolar macrophages (47). We also noted abundant Fas Clearly, understanding the events that control fibroblast and myo- expression by cells located in lymphoid aggregates in IPF patients. fibroblast sensitivity to apoptosis should provide insights into how In contrast, Fas was rarely detected in fibroblastic cells located in these cells persist in the lungs of IPF patients and, desirably, how fibroblast foci. Recently, we also showed that unlike the overlying they may be therapeutically eliminated. Approaches aimed at in- alveolar epithelium, fibroblastic cells within fibroblast foci exhibit creasing fibroblast Fas expression might therefore be therapeuti- minimal nuclear translocation of NF-kB (13). Taken together with cally relevant. the present finding that increased Fas expression by fibroblasts is Downloaded from dependent on NF-kB activation, it is possible that the failure of Acknowledgments fibroblastic cells to undergo apoptosis in fibroblast foci is due in We thank Linda Remigio and Jane Parr for outstanding technical assistance. part to reduced NF-kB activation. In turn, reduced NF-kB acti- We also thank Dr. Doug Everett-Curran (Division of Biostatistics and Bio- vation may contribute to the low level of Fas expression, thereby informatics, National Jewish Health) for advice on the statistical analysis of preventing the ability of these cells to undergo apoptosis. Al- the data. though many mechanisms could contribute to reduced NF-kB http://www.jimmunol.org/ activation, the microarray data presented in this study revealed Disclosures similar patterns of cytokine-induced increases in NF-kB–de- The authors have no financial conflicts of interest. pendent gene expression in primary cultures of normal and fibrotic lung fibroblasts. Furthermore, we previously showed that normal References and fibrotic lung fibroblasts respond to TNF-a and IFN-g in vitro 1. Selman, M., T. E. King, and A. Pardo; American Thoracic Society; European with similar increases in Fas expression (20). In addition, we have Respiratory Society; American College of Chest Physicians. 2001. Idiopathic shown that TGF-b has no effect on the TNF-a and IFN-g in- pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann. Intern. Med. 134: 136–151. duction of Fas expression (20), suggesting that the TGF-b–rich

2. Katzenstein, A. L., and J. L. Myers. 1998. Idiopathic pulmonary fibrosis: clinical by guest on October 1, 2021 environment of the lung parenchyma in IPF has little or no impact relevance of pathologic classification. Am. J. Respir. Crit. Care Med. 157: 1301– on the ability of fibroblasts to increase Fas expression upon ex- 1315. 3. Myers, J. L., and A. L. Katzenstein. 1988. Epithelial necrosis and alveolar posure to the appropriate stimuli. Thus, it is plausible that the collapse in the pathogenesis of usual interstitial pneumonia. Chest 94: 1309– failure to activate NF-kB and increase Fas expression in fibroblast 1311. 4. Hashimoto, N., H. Jin, T. Liu, S. W. Chensue, and S. H. Phan. 2004. Bone foci is due to the absence or reduced presence of the appropriate marrow-derived progenitor cells in pulmonary fibrosis. J. Clin. Invest. 113: 243– NF-kB–activating or other sensitizing stimuli within the fibro- 252. blastic foci. Consistent with this notion, previous studies have 5. Willis, B. C., J. M. Liebler, K. Luby-Phelps, A. G. Nicholson, E. D. Crandall, R. M. du Bois, and Z. Borok. 2005. Induction of epithelial-mesenchymal tran- suggested relative deficiencies of key sensitizing molecules in- sition in alveolar epithelial cells by transforming growth factor-beta1: potential cluding TNF-a, IFN-g, and PGE2 in lung tissues and bron- role in idiopathic pulmonary fibrosis. Am. J. Pathol. 166: 1321–1332. choalveolar lavage specimens from IPF patients (24, 25, 48, 49). 6. Wu, Z., L. Yang, L. Cai, M. Zhang, X. Cheng, X. Yang, and J. Xu. 2007. De- tection of epithelial to mesenchymal transition in airways of a bleomycin in- Taken together, our findings suggest a previously unrecognized duced pulmonary fibrosis model derived from an alpha-smooth muscle actin-Cre role for increased cell-surface Fas expression in promoting the transgenic mouse. Respir. Res. 8: 1. 7. Kim, K. K., M. C. Kugler, P. J. Wolters, L. Robillard, M. G. Galvez, sensitization of lung fibroblasts to apoptosis. This finding obviously A. N. Brumwell, D. Sheppard, and H. A. Chapman. 2006. Alveolar epithelial cell raises the question of the relevant source(s) of FasL. Constitutive mesenchymal transition develops in vivo during pulmonary fibrosis and is reg- cell-surface FasL expression has been detected in a variety of ulated by the extracellular matrix. Proc. Natl. Acad. Sci. USA 103: 13180–13185. 8. Tanjore, H., X. C. Xu, V. V. Polosukhin, A. L. Degryse, B. Li, W. Han, airway epithelial cells including Clara cells (50, 51). Soluble FasL T. P. Sherrill, D. Plieth, E. G. Neilson, T. S. Blackwell, and W. E. Lawson. 2009. has also been detected in the bronchoalveolar lavage fluid of Contribution of epithelial-derived fibroblasts to bleomycin-induced lung fibrosis. patients with a variety of interstitial lung diseases including IPF Am. J. Respir. Crit. Care Med. 180: 657–665. 9. Tomasek, J. J., G. Gabbiani, B. Hinz, C. Chaponnier, and R. A. Brown. 2002. (52, 53). Previous studies have shown FasL to be expressed Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat. by monocytes, macrophages, and NK cells (54, 55), and recent Rev. Mol. Cell Biol. 3: 349–363. 10. Zhang, K., M. D. Rekhter, D. Gordon, and S. H. Phan. 1994. Myofibroblasts and studies have suggested that myofibroblasts themselves are an their role in lung collagen gene expression during pulmonary fibrosis. A com- important source of cell-surface FasL (22, 23). Myofibroblast bined immunohistochemical and in situ hybridization study. Am. J. Pathol. 145: expression of FasL has been implicated in epithelial injury in mice 114–125. 11. Gabbiani, G. 2003. The myofibroblast in wound healing and fibrocontractive following intratracheal instillation with bleomycin (22, 56), and diseases. J. Pathol. 200: 500–503. epithelial cell apoptosis in IPF patients is often seen in areas 12. Lappi-Blanco, E., Y. Soini, and P. Pa¨a¨kko¨. 1999. Apoptotic activity is increased overlying fibroblastic foci (56). Thus, it is conceivable that FasL in the newly formed fibromyxoid connective tissue in bronchiolitis obliterans organizing pneumonia. Lung 177: 367–376. may be constitutively available to engage Fas on fibroblasts and 13. Cha, S. I., S. D. Groshong, S. K. Frankel, B. L. Edelman, G. P. Cosgrove, myofibroblasts. When Fas is expressed at comparatively low J. L. Terry-Powers, L. K. Remigio, D. Curran-Everett, K. K. Brown, C. D. Cool, and D. W. Riches. 2010. Compartmentalized expression of c-FLIP in lung tissues levels, Fas ligation has been shown to stimulate a variety of of patients with idiopathic pulmonary fibrosis. Am. J. Respir. Cell Mol. Biol. 42: nonapoptotic responses including proliferation, survival, tumor 140–148. The Journal of Immunology 537

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Legends for Supplementary Online Materials.

Figure S1. SB203580 and Bay 11-7082 inhibit of p38mapk and NF-κB in lung fibroblastic cells. (A) The p38mapk inhibitor SB203580 (20 μM) blocks both basal, and TNF-α (10 ng/ml) and IFN-γ (50 U/ml) induced phosphorylation of

HSP27, a downstream target of p38mapk, in the human MRC-5 lung fibroblastic cells. Equal loading of protein was verified by blotting for β-actin. The upper panel is a representative blot from one of three independent experiments, while the lower panel shows the mean±SEM for all three experiments. (B) The NF-κB inhibitor Bay 11-7082 (3 μM) blocks TNF-α and IFN-γ induced degradation of

IκB-α in the MRC-5 fibroblasts. Equality in protein loading is illustrated by the β- actin blot. The upper panel is a representative blot from one of three independent experiments, while the lower panel displays the mean ±SEM for all three experiments. The untreated, without vehicle, control is labeled Utr.

Table S1. Transcripts differentially expressed in fibroblast cell lines treated with TNF-α

(10 ng/ml) and IFN-γ (50 U/ml) for 36 hr. These 603 transcripts meet the statistical criteria for differential expression (unpaired t-test, FDR<0.05). The fold ratio

(stimulated/unstimulated) for each fibroblast cell line is given along with the associated p-value for the group.

1 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 203915_at CXCL9 5360.6 5251.0 5104.1 4.49E-09 chemokine (C-X-C motif) ligand 9 210029_at IDO1 4745.4 4316.5 4080.0 1.21E-08 indoleamine 2,3-dioxygenase 1 205890_s_at GABBR1 4318.1 3644.9 4808.9 4.58E-08 gamma-aminobutyric acid (GABA) B receptor, 1 /// UBD /// ubiquitin D 211122_s_at CXCL11 2409.2 2738.5 2224.0 1.42E-07 chemokine (C-X-C motif) ligand 11 1405_i_at CCL5 2010.2 2818.3 2381.2 1.69E-07 chemokine (C-C motif) ligand 5 228501_at GALNTL2 0.034 0.030 0.032 2.53E-07 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase

1555759_a_at CCL5 4927.9 3794.2 5586.6 4.73E-07 chemokine (C-C motif) ligand 5 238581_at GBP5 280.1 283.0 245.0 7.01E-07 guanylate binding protein 5 239206_at CR1L 11.7 14.3 12.4 1.11E-06 complement component (3b/4b) receptor 1-like

239205_s_at CR1 /// 31.5 27.9 34.6 1.20E-06 complement component (3b/4b) receptor 1 CR1L (Knops blood group) /// complement compo 230178_s_at --- 0.201 0.199 0.216 1.74E-06 --- 33304_at ISG20 808.6 823.8 884.5 1.90E-06 interferon stimulated exonuclease gene 20kDa

242625_at RSAD2 894.2 589.6 1355.3 2.87E-06 radical S-adenosyl methionine domain containing 2 226609_at DCBLD1 3.8 4.6 4.2 3.06E-06 discoidin, CUB and LCCL domain containing 1

217933_s_at LAP3 14.6 14.5 16.9 3.29E-06 leucine aminopeptidase 3 224597_at --- 0.421 0.423 0.414 3.38E-06 --- 213797_at RSAD2 1298.2 552.6 1167.5 3.57E-06 radical S-adenosyl methionine domain containing 2 225162_at SH3D19 0.082 0.100 0.107 3.76E-06 SH3 domain containing 19 215485_s_at ICAM1 197.4 238.3 119.2 3.91E-06 intercellular adhesion molecule 1 204655_at CCL5 3474.2 1772.9 3855.3 4.35E-06 chemokine (C-C motif) ligand 5 238455_at --- 0.127 0.109 0.145 4.75E-06 --- 210163_at CXCL11 1358.7 2347.8 1134.0 4.91E-06 chemokine (C-X-C motif) ligand 11 207181_s_at CASP7 4.2 4.0 4.5 5.04E-06 caspase 7, apoptosis-related cysteine peptidase

222868_s_at IL18BP 317.2 320.3 356.3 5.24E-06 interleukin 18 binding protein 202902_s_at CTSS 459.6 664.0 815.1 5.44E-06 cathepsin S 219684_at RTP4 138.1 126.7 174.3 5.46E-06 receptor (chemosensory) transporter protein 4

204698_at ISG20 1046.9 1060.1 1436.9 5.53E-06 interferon stimulated exonuclease gene 20kDa

201980_s_at RSU1 2.7 2.8 2.9 5.62E-06 Ras suppressor protein 1 220358_at BATF3 81.8 124.9 88.6 6.13E-06 basic leucine zipper transcription factor, ATF-like 3

225483_at VPS26B 0.336 0.302 0.334 6.27E-06 vacuolar protein sorting 26 homolog B (S. pombe)

210190_at STX11 7.5 7.1 6.0 6.38E-06 syntaxin 11 230966_at IL4I1 74.3 92.4 122.0 6.61E-06 interleukin 4 induced 1 2 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 202901_x_at CTSS 110.7 135.2 83.8 6.74E-06 cathepsin S 224225_s_at ETV7 61.3 36.0 58.7 6.96E-06 ets variant 7 209545_s_at RIPK2 18.3 20.1 22.0 7.54E-06 receptor-interacting serine-threonine kinase 2

232617_at CTSS 294.9 254.3 433.5 7.65E-06 cathepsin S 204224_s_at GCH1 603.1 831.5 520.0 8.24E-06 GTP cyclohydrolase 1 239582_at PML 15.0 13.6 20.1 8.49E-06 promyelocytic leukemia 219357_at GTPBP1 6.0 7.0 7.5 8.50E-06 GTP binding protein 1 200878_at EPAS1 0.177 0.137 0.148 8.53E-06 endothelial PAS domain protein 1 1555515_a_at C1orf2 5.0 6.4 5.7 8.55E-06 chromosome 1 open reading frame 2 212933_x_at RPL13 0.605 0.629 0.592 9.00E-06 ribosomal protein L13 205660_at OASL 403.5 255.8 602.7 9.75E-06 2'-5'-oligoadenylate synthetase-like 204748_at PTGS2 30.2 23.7 27.7 1.06E-05 prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxyge 224626_at SLC35A4 2.2 2.2 2.4 1.10E-05 solute carrier family 35, member A4 222905_s_at TMEM143 0.540 0.509 0.525 1.13E-05 transmembrane protein 143 1569030_s_at NUB1 5.0 6.5 6.5 1.15E-05 negative regulator of ubiquitin-like proteins 1

209544_at RIPK2 13.2 14.0 13.7 1.16E-05 receptor-interacting serine-threonine kinase 2

212818_s_at ASB1 0.150 0.172 0.124 1.20E-05 ankyrin repeat and SOCS box-containing 1 202590_s_at PDK2 0.204 0.265 0.232 1.22E-05 pyruvate dehydrogenase kinase, isozyme 2 227677_at JAK3 27.5 20.1 35.9 1.25E-05 Janus kinase 3 (a protein tyrosine kinase, leukocyte) 235574_at GBP4 179.5 294.6 135.0 1.37E-05 guanylate binding protein 4 204211_x_at EIF2AK2 3.2 2.7 2.9 1.51E-05 eukaryotic translation initiation factor 2-alpha kinase 2 219716_at APOL6 19.7 12.4 13.4 1.58E-05 apolipoprotein L, 6 209761_s_at SP110 7.2 9.5 10.1 1.58E-05 SP110 nuclear body protein 204683_at ICAM2 13.1 14.1 10.2 1.62E-05 intercellular adhesion molecule 2 217739_s_at NAMPT 7.4 7.8 10.5 1.65E-05 nicotinamide phosphoribosyltransferase 212217_at PREPL 0.244 0.258 0.277 1.68E-05 prolyl endopeptidase-like 219950_s_at TIAM2 1.0 1.0 1.0 1.69E-05 T-cell lymphoma invasion and metastasis 2 200905_x_at HLA-E 6.3 6.4 5.7 1.71E-05 major histocompatibility complex, class I, E 216841_s_at SOD2 29.0 25.8 36.1 1.72E-05 superoxide dismutase 2, mitochondrial 223776_x_at TINF2 5.3 4.4 5.2 1.77E-05 TERF1 (TRF1)-interacting nuclear factor 2 228690_s_at NDUFA11 0.500 0.550 0.541 1.84E-05 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 11, 14.7kDa 215223_s_at SOD2 79.1 57.5 88.5 1.96E-05 superoxide dismutase 2, mitochondrial 228439_at BATF2 194.5 144.1 157.7 1.99E-05 basic leucine zipper transcription factor, ATF-like 2

1559530_at --- 4.5 5.1 4.4 2.02E-05 --- 212505_s_at KIAA0892 1.9 1.9 2.1 2.07E-05 KIAA0892 212733_at KIAA0226 4.6 5.2 6.5 2.07E-05 KIAA0226 58916_at KCTD14 129.2 81.0 87.3 2.09E-05 potassium channel tetramerisation domain containing 14 205599_at TRAF1 209.8 85.6 126.8 2.09E-05 TNF receptor-associated factor 1 3 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 202644_s_at TNFAIP3 135.5 286.1 146.6 2.16E-05 tumor necrosis factor, alpha-induced protein 3

201891_s_at B2M 1.6 1.6 1.5 2.16E-05 beta-2-microglobulin 243176_at --- 0.360 0.318 0.388 2.16E-05 --- AFFX-HUMISGF3STAT1 3.5 3.4 3.8 2.21E-05 signal transducer and activator of transcription 1, 91kDa 224738_x_at RPL7L1 0.487 0.524 0.484 2.23E-05 ribosomal protein L7-like 1 38241_at BTN3A3 10.3 7.4 9.4 2.28E-05 butyrophilin, subfamily 3, member A3 226865_at --- 0.074 0.098 0.068 2.34E-05 --- 219202_at RHBDF2 26.3 31.9 22.2 2.39E-05 rhomboid 5 homolog 2 (Drosophila) 201649_at UBE2L6 5.7 6.3 7.1 2.41E-05 ubiquitin-conjugating enzyme E2L 6 238025_at MLKL 30.4 21.5 31.1 2.41E-05 mixed lineage kinase domain-like 208771_s_at LTA4H 0.311 0.318 0.337 2.44E-05 leukotriene A4 hydrolase 219590_x_at DPH5 0.457 0.419 0.442 2.50E-05 DPH5 homolog (S. cerevisiae) 229625_at GBP5 483.2 667.5 250.3 2.55E-05 guanylate binding protein 5 224452_s_at MGC12966 0.602 0.620 0.561 2.66E-05 hypothetical protein LOC84792

211588_s_at LOC652346 14.0 9.5 11.2 2.67E-05 similar to promyelocytic leukemia protein isoform /// PML 1 /// promyelocytic leukemia 213413_at STON1 0.048 0.032 0.037 2.67E-05 stonin 1 202643_s_at TNFAIP3 336.5 615.2 249.8 2.73E-05 tumor necrosis factor, alpha-induced protein 3

229699_at LOC100129 0.148 0.126 0.114 2.74E-05 Hypothetical protein LOC100129550 550 (LOC100129550), mRNA 208848_at ADH5 0.373 0.436 0.380 2.78E-05 alcohol dehydrogenase 5 (class III), chi polypeptide 212017_at FAM168B 0.174 0.141 0.142 2.81E-05 family with sequence similarity 168, member B

218776_s_at TMEM62 10.4 15.1 17.9 2.92E-05 transmembrane protein 62 203828_s_at IL32 523.8 965.5 1231.9 2.92E-05 interleukin 32 227918_s_at ZYG11B 0.420 0.390 0.442 3.05E-05 zyg-11 homolog B (C. elegans) 204858_s_at TYMP 86.5 47.1 73.8 3.09E-05 thymidine phosphorylase 210797_s_at OASL 363.0 158.6 420.4 3.09E-05 2'-5'-oligoadenylate synthetase-like 219424_at EBI3 58.7 109.6 115.4 3.17E-05 Epstein-Barr virus induced 3 202741_at PRKACB 0.257 0.254 0.260 3.20E-05 protein kinase, cAMP-dependent, catalytic, beta

226757_at IFIT2 49.1 59.7 111.4 3.29E-05 interferon-induced protein with tetratricopeptide repeats 2 223085_at RNF19A 2.2 1.9 2.3 3.35E-05 ring finger protein 19A 32069_at N4BP1 4.9 4.8 5.0 3.37E-05 NEDD4 binding protein 1 233540_s_at CDK5RAP2 0.274 0.342 0.272 3.42E-05 CDK5 regulatory subunit associated protein 2

240214_at --- 0.505 0.530 0.501 3.43E-05 --- 206553_at OAS2 23.0 43.2 30.3 3.46E-05 2'-5'-oligoadenylate synthetase 2, 69/71kDa 225906_at --- 0.059 0.080 0.092 3.53E-05 --- 217982_s_at MORF4L1 0.770 0.768 0.802 3.58E-05 mortality factor 4 like 1 214459_x_at HLA-C 3.1 3.0 3.6 3.62E-05 major histocompatibility complex, class I, C 4 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 1553293_at MRGPRX3 368.4 152.8 317.1 3.63E-05 MAS-related GPR, member X3 238226_at FAM70B 2.4 2.0 2.2 3.91E-05 family with sequence similarity 70, member B

202727_s_at IFNGR1 1.9 1.9 2.2 3.93E-05 interferon gamma receptor 1 226233_at B3GALNT2 0.330 0.265 0.338 4.00E-05 beta-1,3-N-acetylgalactosaminyltransferase 2

219181_at LIPG 13.1 13.8 20.6 4.09E-05 lipase, endothelial 208812_x_at HLA-C 3.0 2.6 2.7 4.23E-05 major histocompatibility complex, class I, C 203927_at NFKBIE 26.4 28.4 40.3 4.24E-05 nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, ep 225338_at ZYG11B 0.282 0.325 0.366 4.26E-05 zyg-11 homolog B (C. elegans) 235964_x_at --- 12.9 15.0 15.7 4.44E-05 --- 217986_s_at BAZ1A 5.8 6.2 7.2 4.51E-05 bromodomain adjacent to zinc finger domain, 1A

201339_s_at SCP2 0.456 0.426 0.386 4.53E-05 sterol carrier protein 2 204785_x_at IFNAR2 4.3 3.8 5.2 4.60E-05 interferon (alpha, beta and omega) receptor 2

213620_s_at ICAM2 40.1 24.2 52.7 4.61E-05 intercellular adhesion molecule 2 1553118_at THEM4 0.394 0.373 0.430 4.62E-05 thioesterase superfamily member 4 202687_s_at TNFSF10 436.7 546.5 1171.7 4.64E-05 tumor necrosis factor (ligand) superfamily, member 10 208745_at ATP5L 0.495 0.428 0.487 4.66E-05 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit G 220751_s_at C5orf4 0.065 0.097 0.085 4.70E-05 chromosome 5 open reading frame 4 204533_at CXCL10 4062.9 1500.4 5830.3 4.70E-05 chemokine (C-X-C motif) ligand 10 214329_x_at TNFSF10 294.0 190.9 376.2 4.73E-05 tumor necrosis factor (ligand) superfamily, member 10 205552_s_at OAS1 327.7 271.0 728.0 5.11E-05 2',5'-oligoadenylate synthetase 1, 40/46kDa 212605_s_at --- 0.259 0.205 0.173 5.12E-05 --- 201857_at ZFR 0.669 0.711 0.717 5.17E-05 zinc finger RNA binding protein 217738_at NAMPT 8.1 7.7 12.7 5.24E-05 nicotinamide phosphoribosyltransferase 226833_at CYB5D1 0.327 0.388 0.378 5.26E-05 cytochrome b5 domain containing 1 202136_at ZMYND11 0.427 0.378 0.329 5.30E-05 zinc finger, MYND domain containing 11 229174_at C3orf38 5.1 5.4 5.9 5.39E-05 chromosome 3 open reading frame 38 240013_at --- 7.8 7.3 11.1 5.41E-05 --- 212741_at MAOA 0.047 0.058 0.059 5.50E-05 monoamine oxidase A 238007_at ZNF271 0.451 0.394 0.386 5.51E-05 zinc finger protein 271 203607_at INPP5F 0.446 0.423 0.514 5.54E-05 inositol polyphosphate-5-phosphatase F 208073_x_at TTC3 0.126 0.129 0.118 5.55E-05 tetratricopeptide repeat domain 3 202108_at PEPD 0.598 0.621 0.659 5.58E-05 peptidase D 200817_x_at RPS10 0.573 0.612 0.571 5.64E-05 ribosomal protein S10 204019_s_at SH3YL1 0.173 0.188 0.212 5.70E-05 SH3 domain containing, Ysc84-like 1 (S. cerevisiae)

209619_at CD74 740.5 242.9 1351.8 5.76E-05 CD74 molecule, major histocompatibility complex, class II invariant chain 214440_at NAT1 2.5 2.2 2.6 5.78E-05 N-acetyltransferase 1 (arylamine N- acetyltransferase) 5 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 213138_at ARID5A 6.9 6.3 9.2 5.98E-05 AT rich interactive domain 5A (MRF1-like) 225729_at C6orf89 0.283 0.335 0.345 6.05E-05 chromosome 6 open reading frame 89 229653_at VPS53 0.326 0.330 0.309 6.07E-05 vacuolar protein sorting 53 homolog (S. cerevisiae) 204389_at MAOA 0.129 0.100 0.160 6.18E-05 monoamine oxidase A 204187_at GMPR 23.4 23.4 40.8 6.27E-05 guanosine monophosphate reductase 202688_at TNFSF10 1057.9 1084.1 1012.8 6.27E-05 tumor necrosis factor (ligand) superfamily, member 10 207375_s_at IL15RA 55.7 39.5 94.2 6.28E-05 interleukin 15 receptor, alpha 224763_at RPL37 0.189 0.199 0.216 6.57E-05 Ribosomal protein L37, mRNA (cDNA clone IMAGE:6671394) 225216_at CXorf39 0.411 0.436 0.357 6.61E-05 CDNA: FLJ21191 fis, clone COL00104 235505_s_at --- 0.139 0.185 0.172 6.68E-05 --- 222582_at PRKAG2 0.357 0.330 0.357 6.73E-05 protein kinase, AMP-activated, gamma 2 non- catalytic subunit 208012_x_at SP110 7.9 8.9 12.4 6.83E-05 SP110 nuclear body protein 219634_at CHST11 4.0 4.9 5.6 6.84E-05 carbohydrate (chondroitin 4) sulfotransferase 11

204249_s_at LMO2 26.9 51.9 32.9 6.94E-05 LIM domain only 2 (rhombotin-like 1) 225123_at --- 0.075 0.046 0.087 7.19E-05 --- 235529_x_at --- 15.4 14.2 15.9 7.20E-05 --- 202307_s_at TAP1 25.7 29.8 42.4 7.20E-05 transporter 1, ATP-binding cassette, sub-family B (MDR/TAP) 232666_at OAS3 18.6 35.7 18.8 7.23E-05 2'-5'-oligoadenylate synthetase 3, 100kDa 203652_at MAP3K11 1.6 1.8 1.6 7.33E-05 mitogen-activated protein kinase kinase kinase 11

210538_s_at BIRC3 133.0 134.4 127.3 7.56E-05 baculoviral IAP repeat-containing 3 229553_at PGM2L1 0.427 0.421 0.408 7.57E-05 phosphoglucomutase 2-like 1 226551_at RIPK1 2.9 3.9 3.5 7.58E-05 receptor (TNFRSF)-interacting serine-threonine kinase 1 219809_at WDR55 2.5 2.4 2.3 7.62E-05 WD repeat domain 55 201076_at NHP2L1 0.563 0.611 0.575 7.66E-05 NHP2 non-histone chromosome protein 2-like 1 (S. cerevisiae) 201814_at TBC1D5 0.483 0.433 0.464 7.68E-05 TBC1 domain family, member 5 225414_at RNF149 3.3 3.9 4.4 7.72E-05 ring finger protein 149 225944_at NLN 0.328 0.383 0.418 7.80E-05 neurolysin (metallopeptidase M3 family) 224809_x_at TINF2 5.7 4.2 5.4 7.87E-05 TERF1 (TRF1)-interacting nuclear factor 2 235170_at ZNF92 2.1 1.8 1.8 7.94E-05 zinc finger protein 92 239246_at FARP1 0.179 0.225 0.205 7.98E-05 CDEP 213932_x_at HLA-A 2.3 2.6 2.4 8.13E-05 major histocompatibility complex, class I, A 238029_s_at SLC16A14 0.071 0.046 0.085 8.31E-05 solute carrier family 16, member 14 (monocarboxylic acid transporter 14) 209762_x_at SP110 7.8 9.9 12.4 8.48E-05 SP110 nuclear body protein 225625_at ALKBH2 0.450 0.496 0.434 8.53E-05 alkB, alkylation repair homolog 2 (E. coli) 223192_at SLC25A28 7.8 10.7 13.9 8.66E-05 solute carrier family 25, member 28 213716_s_at SECTM1 34.8 36.2 97.0 8.91E-05 secreted and transmembrane 1 1552323_s_at FAM122C 12.4 17.3 11.3 8.98E-05 family with sequence similarity 122C 6 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 222572_at PPM2C 2.2 2.3 2.1 9.35E-05 protein phosphatase 2C, magnesium-dependent, catalytic subunit 217780_at C19orf56 0.668 0.678 0.739 9.41E-05 chromosome 19 open reading frame 56 226474_at NLRC5 17.3 21.7 18.8 9.46E-05 NLR family, CARD domain containing 5 204999_s_at ATF5 8.4 10.7 11.7 9.54E-05 activating transcription factor 5 211678_s_at RNF114 4.2 5.0 5.7 9.63E-05 ring finger protein 114 211528_x_at HLA-G 4.2 3.7 4.5 9.63E-05 major histocompatibility complex, class I, G 220744_s_at IFT122 0.424 0.496 0.494 9.73E-05 intraflagellar transport 122 homolog (Chlamydomonas) 225228_at TMEM77 0.710 0.713 0.772 9.83E-05 transmembrane protein 77 228797_at --- 0.245 0.181 0.245 9.84E-05 --- 231779_at IRAK2 21.9 23.0 15.7 9.92E-05 interleukin-1 receptor-associated kinase 2 231137_at --- 0.390 0.461 0.450 9.99E-05 --- 226883_at --- 0.358 0.338 0.282 9.99E-05 --- 217947_at CMTM6 0.728 0.667 0.709 1.00E-04 CKLF-like MARVEL transmembrane domain containing 6 205606_at LRP6 0.225 0.228 0.186 0.0001 low density lipoprotein receptor-related protein 6

235175_at GBP4 208.8 96.3 246.9 0.0001 guanylate binding protein 4 216526_x_at HLA-C 2.5 2.4 3.0 0.0001 major histocompatibility complex, class I, C 210785_s_at C1orf38 29.4 30.5 42.2 0.0001 chromosome 1 open reading frame 38 203137_at WTAP 5.2 4.2 6.5 0.0001 Wilms tumor 1 associated protein 201422_at IFI30 67.0 159.6 295.9 0.00011 interferon, gamma-inducible protein 30 201962_s_at RNF41 0.279 0.256 0.190 0.00011 ring finger protein 41 204821_at BTN3A3 8.8 7.2 9.7 0.00011 butyrophilin, subfamily 3, member A3 203449_s_at TERF1 0.597 0.627 0.658 0.00011 telomeric repeat binding factor (NIMA- interacting) 1 205236_x_at SOD3 12.3 6.5 10.6 0.00011 superoxide dismutase 3, extracellular 217752_s_at CNDP2 4.7 4.1 5.0 0.00011 CNDP dipeptidase 2 (metallopeptidase M20 family) 209124_at MYD88 6.7 4.7 6.8 0.00011 myeloid differentiation primary response gene (88) 219863_at HERC5 61.1 72.1 142.3 0.00011 hect domain and RLD 5 201317_s_at PSMA2 2.5 3.2 2.7 0.00011 proteasome (prosome, macropain) subunit, alpha type, 2 209815_at PTCH1 0.158 0.085 0.090 0.00011 patched homolog 1 (Drosophila) 219283_at C1GALT1C 6.5 4.9 6.5 0.00011 C1GALT1-specific chaperone 1 1 212993_at NACC2 0.411 0.381 0.317 0.00011 NACC family member 2, BEN and BTB (POZ) domain containing 232021_at GLT8D3 0.451 0.486 0.425 0.00011 glycosyltransferase 8 domain containing 3 243141_at SGMS2 0.160 0.106 0.126 0.00011 sphingomyelin synthase 2 203550_s_at C1orf2 3.8 4.3 5.7 0.00011 chromosome 1 open reading frame 2 204439_at IFI44L 230.8 115.7 690.6 0.00012 interferon-induced protein 44-like 226395_at HOOK3 0.272 0.230 0.270 0.00012 hook homolog 3 (Drosophila) 211529_x_at HLA-G 4.0 5.3 4.8 0.00012 major histocompatibility complex, class I, G 7 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 206272_at RAB4A /// 0.171 0.227 0.267 0.00012 RAB4A, member RAS oncogene family /// S-phase SPHAR response (cyclin-related) 200887_s_at STAT1 2.9 2.5 3.2 0.00012 signal transducer and activator of transcription 1, 91kDa 220975_s_at C1QTNF1 16.0 15.0 21.2 0.00012 C1q and tumor necrosis factor related protein 1

206065_s_at DPYS 1.6 1.4 1.4 0.00012 dihydropyrimidinase 217502_at IFIT2 210.8 129.3 249.9 0.00012 interferon-induced protein with tetratricopeptide repeats 2 219540_at ZNF267 10.8 9.2 14.7 0.00012 zinc finger protein 267 201064_s_at PABPC4 0.183 0.198 0.168 0.00012 poly(A) binding protein, cytoplasmic 4 (inducible form) 203241_at UVRAG 3.8 3.7 4.7 0.00012 UV radiation resistance associated gene 225199_at --- 0.225 0.222 0.286 0.00012 --- 212163_at KIDINS220 0.431 0.482 0.452 0.00012 kinase D-interacting substrate, 220kDa

200813_s_at PAFAH1B1 0.537 0.507 0.557 0.00012 platelet-activating factor acetylhydrolase, isoform Ib, alpha subunit 45kDa 219691_at SAMD9 7.2 14.1 13.3 0.00012 sterile alpha motif domain containing 9 226502_at ELMOD2 0.369 0.309 0.287 0.00013 ELMO/CED-12 domain containing 2 225468_at PATL1 2.5 3.4 3.2 0.00013 protein associated with topoisomerase II homolog 1 (yeast) 238417_at PGM2L1 0.532 0.535 0.612 0.00013 phosphoglucomutase 2-like 1 210119_at KCNJ15 19.1 23.5 38.1 0.00013 potassium inwardly-rectifying channel, subfamily J, member 15 204804_at TRIM21 7.2 6.1 9.0 0.00013 tripartite motif-containing 21 219545_at KCTD14 83.3 81.2 68.0 0.00013 potassium channel tetramerisation domain containing 14 221827_at RBCK1 3.3 2.8 3.3 0.00013 RanBP-type and C3HC4-type zinc finger containing 1 221731_x_at VCAN 0.155 0.119 0.102 0.00013 versican 226157_at TFDP2 0.106 0.132 0.103 0.00013 CDNA clone IMAGE:4817695 219210_s_at RAB8B 4.8 5.4 4.7 0.00013 RAB8B, member RAS oncogene family 228495_at CCDC75 0.160 0.192 0.163 0.00013 coiled-coil domain containing 75 204806_x_at HLA-F 7.4 7.1 10.7 0.00013 major histocompatibility complex, class I, F 222864_s_at ZNF219 1.0 1.0 1.0 0.00013 zinc finger protein 219 227026_at MPHOSPH 0.526 0.494 0.567 0.00014 M-phase phosphoprotein 8 8 226175_at TTC9C 3.6 3.1 3.7 0.00014 tetratricopeptide repeat domain 9C 205486_at TESK2 6.0 6.0 7.7 0.00014 testis-specific kinase 2 216862_s_at LOC100133 0.232 0.333 0.266 0.00014 similar to mature T-cell proliferations 1 /// mature 946 /// T-cell proliferation 1 MTCP1 222288_at --- 0.049 0.026 0.026 0.00014 --- 218611_at IER5 1.8 1.7 1.8 0.00014 immediate early response 5 8 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 217436_x_at HLA- 6.0 8.1 9.1 0.00015 major histocompatibility complex, class I, A /// A,A29,B,G, major histocompatibility comple H,J 36129_at SGSM2 0.432 0.498 0.506 0.00015 small G protein signaling modulator 2 226207_at RILPL1 2.7 2.5 3.6 0.00015 Rab interacting lysosomal protein-like 1 204620_s_at VCAN 0.136 0.144 0.138 0.00015 versican 205546_s_at TYK2 2.0 1.9 2.3 0.00015 tyrosine kinase 2 235229_at --- 904.9 328.5 1470.6 0.00015 --- 229673_at --- 0.343 0.294 0.295 0.00015 --- 223834_at CD274 100.1 46.6 59.1 0.00015 CD274 molecule 201429_s_at RPL37A 0.740 0.750 0.794 0.00015 ribosomal protein L37a 224643_at PRRC1 0.441 0.420 0.420 0.00015 proline-rich coiled-coil 1 221477_s_at SOD2 15.0 19.8 32.6 0.00016 superoxide dismutase 2, mitochondrial 1555736_a_at AGTRAP 3.0 3.8 3.1 0.00016 angiotensin II receptor-associated protein 1554997_a_at PTGS2 20.0 20.0 11.0 0.00016 prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxyge 209770_at BTN3A1 6.7 7.3 8.1 0.00016 butyrophilin, subfamily 3, member A1 229351_at --- 0.654 0.686 0.613 0.00016 --- 200012_x_at LOC653737 0.449 0.460 0.421 0.00016 hypothetical LOC653737 /// ribosomal protein L21-like /// ribosomal protein L21 55692_at ELMO2 5.0 5.5 7.7 0.00017 engulfment and cell motility 2 204084_s_at CLN5 2.3 2.3 2.8 0.00017 ceroid-lipofuscinosis, neuronal 5 204303_s_at KIAA0427 0.246 0.168 0.228 0.00017 KIAA0427 208617_s_at PTP4A2 0.509 0.436 0.450 0.00017 protein tyrosine phosphatase type IVA, member 2

211506_s_at IL8 539.6 2763.7 689.5 0.00017 interleukin 8 210563_x_at CFLAR 7.8 9.4 10.4 0.00017 CASP8 and FADD-like apoptosis regulator 240054_at --- 12.3 10.5 6.6 0.00017 --- 208392_x_at SP110 13.7 11.0 25.1 0.00017 SP110 nuclear body protein 225376_at C20orf11 0.451 0.402 0.519 0.00017 chromosome 20 open reading frame 11 226656_at CRTAP 0.363 0.274 0.253 0.00017 cartilage associated protein 212432_at GRPEL1 1.5 1.6 1.8 0.00017 GrpE-like 1, mitochondrial (E. coli) 202859_x_at IL8 145.7 777.2 333.8 0.00017 interleukin 8 219013_at GALNT11 0.189 0.241 0.239 0.00017 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase

233090_at --- 0.474 0.422 0.488 0.00018 --- 204098_at RBMX2 0.668 0.620 0.688 0.00018 RNA binding motif protein, X-linked 2 222916_s_at HDLBP 0.187 0.208 0.130 0.00018 high density lipoprotein binding protein 238531_x_at --- 111.1 61.2 238.3 0.00018 --- 1559433_at LOC149773 5.1 8.2 7.0 0.00018 CDNA FLJ33293 fis, clone BNGH42000675

226009_at RP11- 0.277 0.394 0.349 0.00018 deleted in a mouse model of primary ciliary 529I10.4 dyskinesia 225973_at TAP2 14.4 12.8 20.9 0.00018 transporter 2, ATP-binding cassette, sub-family B (MDR/TAP) 9 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 203552_at MAP4K5 0.438 0.408 0.351 0.00019 mitogen-activated protein kinase kinase kinase kinase 5 204828_at RAD9A 5.8 9.0 7.7 0.00019 RAD9 homolog A (S. pombe) 1556629_a_at SNAP25 0.389 0.409 0.420 0.00019 HUMSNAP25B(F) 218817_at SPCS3 2.2 1.9 2.2 0.00019 signal peptidase complex subunit 3 homolog (S. cerevisiae) 204972_at OAS2 394.1 133.0 498.5 0.00019 2'-5'-oligoadenylate synthetase 2, 69/71kDa 219364_at DHX58 26.9 33.0 24.6 0.00019 DEXH (Asp-Glu-X-His) box polypeptide 58 205692_s_at CD38 149.2 401.4 677.2 0.00019 CD38 molecule 202126_at PRPF4B 1.5 1.3 1.5 0.00019 PRP4 pre-mRNA processing factor 4 homolog B (yeast) 221253_s_at TXNDC5 0.549 0.460 0.478 0.00019 thioredoxin domain containing 5 208453_s_at XPNPEP1 2.6 3.3 3.1 0.00019 X-prolyl aminopeptidase (aminopeptidase P) 1, soluble 206633_at CHRNA1 30.4 31.7 86.3 0.0002 cholinergic receptor, nicotinic, alpha 1 (muscle)

203236_s_at LGALS9 45.7 109.9 144.8 0.0002 lectin, galactoside-binding, soluble, 9 207630_s_at CREM 6.0 4.8 4.9 0.0002 cAMP responsive element modulator 204070_at RARRES3 47.3 42.7 30.0 0.0002 retinoic acid receptor responder (tazarotene induced) 3 219092_s_at IPPK 2.1 2.0 1.9 0.0002 inositol 1,3,4,5,6-pentakisphosphate 2-kinase

225424_at GPAM 0.146 0.183 0.147 0.0002 glycerol-3-phosphate acyltransferase, mitochondrial 215342_s_at RABGAP1L 2.3 2.6 3.1 0.0002 RAB GTPase activating protein 1-like

226756_at --- 4.1 5.0 4.7 0.0002 --- 217922_at --- 0.088 0.131 0.161 0.00021 --- 214791_at SP140L 2.5 3.4 2.7 0.00021 SP140 nuclear body protein-like 220091_at SLC2A6 29.7 16.9 42.7 0.00021 solute carrier family 2 (facilitated glucose transporter), member 6 204470_at CXCL1 42.9 124.8 94.7 0.00021 chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha)

201115_at POLD2 0.476 0.363 0.430 0.00021 polymerase (DNA directed), delta 2, regulatory subunit 50kDa 235157_at --- 16.9 23.3 28.6 0.00021 --- 235046_at --- 0.186 0.246 0.311 0.00021 --- 226603_at SAMD9L 5.5 10.0 12.2 0.00021 sterile alpha motif domain containing 9-like 231175_at BEND6 0.291 0.268 0.292 0.00021 BEN domain containing 6 227125_at --- 20.0 15.7 21.1 0.00021 --- 204769_s_at TAP2 19.4 15.9 14.8 0.00021 transporter 2, ATP-binding cassette, sub-family B (MDR/TAP) 231296_at --- 0.407 0.372 0.338 0.00021 --- 200704_at LITAF 2.3 2.7 3.0 0.00022 lipopolysaccharide-induced TNF factor 220052_s_at TINF2 4.8 3.8 5.1 0.00022 TERF1 (TRF1)-interacting nuclear factor 2 10 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 224645_at EIF4EBP2 0.221 0.200 0.282 0.00022 eukaryotic translation initiation factor 4E binding protein 2 239218_at PDE1C 0.070 0.078 0.069 0.00022 phosphodiesterase 1C, calmodulin-dependent 70kDa 209203_s_at BICD2 0.449 0.453 0.473 0.00022 bicaudal D homolog 2 (Drosophila) 205875_s_at ATRIP /// 12.8 25.9 39.0 0.00022 ATR interacting protein /// three prime repair TREX1 exonuclease 1 219209_at IFIH1 125.4 49.1 132.5 0.00022 interferon induced with helicase C domain 1

202588_at AK1 0.466 0.371 0.399 0.00022 adenylate kinase 1 225302_at TXNDC10 0.297 0.367 0.312 0.00022 thioredoxin domain containing 10 227270_at LOC285550 0.258 0.299 0.361 0.00022 hypothetical protein LOC285550

218793_s_at SCML1 2.5 3.5 3.0 0.00023 sex comb on midleg-like 1 (Drosophila) 225279_s_at C3orf17 0.514 0.572 0.579 0.00023 chromosome 3 open reading frame 17 222394_at PDCD6IP 0.223 0.276 0.355 0.00023 programmed cell death 6 interacting protein

209239_at NFKB1 4.2 5.7 3.8 0.00023 nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 1570253_a_at RHEBL1 8.3 13.2 7.3 0.00023 Ras homolog enriched in brain like 1 225012_at HDLBP 0.273 0.190 0.267 0.00023 high density lipoprotein binding protein 37549_g_at BBS9 0.517 0.387 0.424 0.00023 Bardet-Biedl syndrome 9 231769_at FBXO6 19.0 15.7 28.1 0.00023 F-box protein 6 221798_x_at --- 0.763 0.785 0.711 0.00023 --- 204747_at IFIT3 56.0 28.1 133.8 0.00023 interferon-induced protein with tetratricopeptide repeats 3 223454_at CXCL16 18.2 33.0 44.7 0.00023 chemokine (C-X-C motif) ligand 16 212206_s_at H2AFV 0.111 0.090 0.105 0.00024 H2A histone family, member V 209774_x_at CXCL2 53.7 51.2 19.3 0.00024 chemokine (C-X-C motif) ligand 2 204494_s_at C15orf39 1.7 1.8 1.6 0.00024 chromosome 15 open reading frame 39 201287_s_at SDC1 0.280 0.163 0.160 0.00024 syndecan 1 212734_x_at RPL13 0.560 0.664 0.561 0.00024 ribosomal protein L13 213151_s_at 7-Sep 0.624 0.532 0.543 0.00024 septin 7 200668_s_at UBE2D3 1.5 1.5 1.4 0.00024 ubiquitin-conjugating enzyme E2D 3 (UBC4/5 homolog, yeast) 224619_at CASC4 0.540 0.617 0.576 0.00025 cancer susceptibility candidate 4 212830_at MEGF9 0.107 0.061 0.071 0.00025 multiple EGF-like-domains 9 221476_s_at RPL15 0.331 0.313 0.349 0.00025 ribosomal protein L15 202251_at PRPF3 3.8 3.7 4.1 0.00025 PRP3 pre-mRNA processing factor 3 homolog (S. cerevisiae) 226047_at MRVI1 0.019 0.058 0.021 0.00025 murine retrovirus integration site 1 homolog

209630_s_at FBXW2 0.298 0.222 0.329 0.00025 F-box and WD repeat domain containing 2 200717_x_at RPL7 0.470 0.543 0.402 0.00025 ribosomal protein L7 207616_s_at TANK 2.2 3.1 2.9 0.00025 TRAF family member-associated NFKB activator

211799_x_at HLA-C 5.3 5.4 5.7 0.00025 major histocompatibility complex, class I, C 11 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 222870_s_at B3GNT2 5.7 4.1 8.1 0.00025 UDP-GlcNAc:betaGal beta-1,3-N- acetylglucosaminyltransferase 2 205299_s_at BTN2A2 4.5 4.8 6.1 0.00025 butyrophilin, subfamily 2, member A2 230214_at MRVI1 0.038 0.065 0.026 0.00025 murine retrovirus integration site 1 homolog

212657_s_at IL1RN 32.6 18.3 45.4 0.00026 interleukin 1 receptor antagonist 224784_at MLLT6 4.0 5.4 5.6 0.00026 myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); tran

235513_at --- 0.231 0.343 0.252 0.00026 --- 227069_at --- 0.180 0.239 0.174 0.00026 --- 225929_s_at RNF213 14.0 12.6 14.1 0.00026 ring finger protein 213 221528_s_at ELMO2 4.9 6.1 9.4 0.00026 engulfment and cell motility 2 222997_s_at MRPS21 0.666 0.754 0.698 0.00026 mitochondrial ribosomal protein S21 227085_at H2AFV 0.410 0.282 0.275 0.00026 H2A histone family, member V 225849_s_at SFT2D1 1.5 1.7 1.7 0.00026 SFT2 domain containing 1 201783_s_at RELA 4.7 4.2 7.8 0.00026 v-rel reticuloendotheliosis viral oncogene homolog A (avian) 203283_s_at HS2ST1 0.418 0.480 0.433 0.00027 heparan sulfate 2-O-sulfotransferase 1 217781_s_at ZFP106 0.204 0.270 0.216 0.00027 zinc finger protein 106 homolog (mouse) 243271_at --- 14.7 27.6 26.7 0.00027 --- 226648_at HIF1AN 0.262 0.268 0.347 0.00027 hypoxia inducible factor 1, alpha subunit inhibitor

217336_at Various' 0.484 0.505 0.601 0.00027 hypothetical LOC376693 /// similar to ribosomal protein S10 /// similar to hCG20 210443_x_at OGFR 3.4 3.9 5.5 0.00027 opioid growth factor receptor 201273_s_at hCG_1781 0.657 0.684 0.712 0.00027 hCG1781062 /// signal recognition particle 9kDa 062 /// SRP9 226501_at XPNPEP3 0.285 0.332 0.233 0.00027 X-prolyl aminopeptidase (aminopeptidase P) 3, putative 228347_at SIX1 4.3 4.2 4.0 0.00028 SIX homeobox 1 201502_s_at NFKBIA 19.7 11.2 35.9 0.00028 nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, al 227221_at --- 0.268 0.250 0.335 0.00028 --- 208860_s_at ATRX 0.273 0.293 0.200 0.00028 alpha thalassemia/mental retardation syndrome X- linked (RAD54 homolog, S. cerevi

241347_at KIAA1618 11.2 12.2 6.7 0.00028 KIAA1618 216231_s_at B2M 1.5 1.8 1.7 0.00028 beta-2-microglobulin 205500_at C5 0.533 0.591 0.525 0.00028 complement component 5 226333_at IL6R 0.173 0.167 0.098 0.00028 interleukin 6 receptor 211542_x_at RPS10 0.594 0.630 0.586 0.00029 ribosomal protein S10 225923_at --- 0.121 0.184 0.152 0.00029 --- 226516_at C19orf28 3.2 3.0 3.1 0.00029 chromosome 19 open reading frame 28 226805_at --- 0.099 0.083 0.082 0.00029 --- 204072_s_at FRY 0.026 0.045 0.024 0.00029 furry homolog (Drosophila) 12 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 212736_at C16orf45 0.205 0.290 0.184 0.00029 chromosome 16 open reading frame 45 210645_s_at TTC3 0.093 0.164 0.101 0.00029 tetratricopeptide repeat domain 3 218213_s_at C11orf10 0.547 0.630 0.589 0.0003 chromosome 11 open reading frame 10 224702_at TMEM167 0.552 0.429 0.434 0.0003 transmembrane protein 167A A 211256_x_at BTN2A1 4.8 4.2 6.5 0.0003 butyrophilin, subfamily 2, member A1 221726_at RPL22 0.246 0.231 0.151 0.0003 ribosomal protein L22 226117_at TIFA 13.6 13.7 12.8 0.0003 TRAF-interacting protein with forkhead-associated domain 215313_x_at HLA-A 2.4 2.4 3.1 0.0003 major histocompatibility complex, class I, A 209417_s_at IFI35 84.4 32.3 44.6 0.0003 interferon-induced protein 35 223220_s_at PARP9 15.8 7.2 18.4 0.0003 poly (ADP-ribose) polymerase family, member 9

205945_at IL6R 0.100 0.108 0.046 0.0003 interleukin 6 receptor 228505_s_at TMEM170 4.7 5.7 3.7 0.0003 transmembrane protein 170A A 219774_at CCDC93 2.6 2.7 3.2 0.00031 coiled-coil domain containing 93 206469_x_at AKR7A3 0.455 0.451 0.522 0.00031 aldo-keto reductase family 7, member A3 (aflatoxin aldehyde reductase) 225617_at ODF2 0.479 0.373 0.432 0.00031 outer dense fiber of sperm tails 2 217751_at GSTK1 4.4 4.0 5.6 0.00031 glutathione S-transferase kappa 1 200790_at ODC1 0.389 0.359 0.287 0.00031 ornithine decarboxylase 1 202906_s_at NBN 4.0 5.5 5.9 0.00031 nibrin 225147_at CYTH3 0.306 0.380 0.457 0.00031 cytohesin 3 208661_s_at TTC3 0.096 0.153 0.121 0.00032 tetratricopeptide repeat domain 3 221953_s_at LOC729580 0.359 0.282 0.333 0.00032 PREDICTED: Homo sapiens hypothetical LOC729580 (LOC729580), mRNA 223598_at RAD23B 0.287 0.397 0.352 0.00032 RAD23 homolog B (S. cerevisiae) 209636_at NFKB2 14.1 18.7 15.5 0.00032 nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (p49/p100) 203299_s_at AP1S2 0.586 0.572 0.478 0.00032 adaptor-related protein complex 1, sigma 2 subunit 201335_s_at ARHGEF12 0.291 0.278 0.251 0.00032 Rho guanine nucleotide exchange factor (GEF) 12

226771_at ATP8B2 0.521 0.449 0.383 0.00032 ATPase, class I, type 8B, member 2 233880_at RNF213 16.9 14.6 13.9 0.00033 ring finger protein 213 219104_at RNF141 0.128 0.234 0.161 0.00033 ring finger protein 141 219563_at C14orf139 0.176 0.252 0.210 0.00033 chromosome 14 open reading frame 139 222455_s_at PARVA 0.663 0.567 0.588 0.00033 parvin, alpha 224769_at --- 0.733 0.627 0.634 0.00033 --- 219593_at SLC15A3 34.9 22.0 67.5 0.00033 solute carrier family 15, member 3 227531_at --- 0.350 0.446 0.289 0.00033 --- 225931_s_at RNF213 14.0 16.0 14.5 0.00033 ring finger protein 213 207571_x_at C1orf38 25.1 21.0 36.8 0.00033 chromosome 1 open reading frame 38 1553982_a_at RAB7B 0.286 0.287 0.322 0.00033 RAB7B, member RAS oncogene family 226512_at ZMYM2 0.401 0.462 0.516 0.00034 zinc finger, MYM-type 2 13 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 208619_at DDB1 0.400 0.514 0.431 0.00034 damage-specific DNA binding protein 1, 127kDa

212042_x_at RPL7 0.469 0.493 0.410 0.00034 ribosomal protein L7 211373_s_at PSEN2 3.8 5.7 4.6 0.00034 presenilin 2 (Alzheimer disease 4) 200867_at RNF114 4.4 6.6 7.4 0.00034 ring finger protein 114 221740_x_at LRRC37A2 0.226 0.265 0.183 0.00034 CDNA FLJ34414 fis, clone HEART2003168, highly similar to Homo sapiens c114 SLIT- 215719_x_at FAS 4.0 6.1 8.1 0.00034 Fas (TNF receptor superfamily, member 6) 217456_x_at HLA-E 4.7 3.9 2.8 0.00034 major histocompatibility complex, class I, E 218130_at C17orf62 3.0 2.7 4.3 0.00034 chromosome 17 open reading frame 62 215602_at FGD2 1.0 1.0 1.0 0.00034 FYVE, RhoGEF and PH domain containing 2 202209_at LSM3 0.605 0.506 0.544 0.00034 LSM3 homolog, U6 small nuclear RNA associated (S. cerevisiae) 233310_at --- 3.8 5.7 5.2 0.00034 --- 234987_at --- 18.6 18.1 14.6 0.00034 --- 201665_x_at RPS17 0.513 0.457 0.482 0.00035 ribosomal protein S17 202381_at ADAM9 0.717 0.781 0.746 0.00035 ADAM metallopeptidase domain 9 (meltrin gamma) 223983_s_at C19orf12 2.4 2.7 3.1 0.00035 chromosome 19 open reading frame 12 214318_s_at FRY 0.269 0.157 0.200 0.00035 furry homolog (Drosophila) 241991_at --- 3.4 4.3 5.3 0.00035 --- 1558102_at --- 0.314 0.312 0.245 0.00035 --- 209878_s_at RELA 6.7 5.5 10.6 0.00035 v-rel reticuloendotheliosis viral oncogene homolog A (avian) 225415_at DTX3L 6.8 4.9 9.4 0.00035 deltex 3-like (Drosophila) 204413_at TRAF2 3.1 4.0 2.8 0.00035 TNF receptor-associated factor 2 218943_s_at DDX58 50.4 35.4 118.8 0.00035 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 221011_s_at LBH 0.152 0.092 0.100 0.00035 limb bud and heart development homolog (mouse) 221680_s_at ETV7 16.3 13.1 20.7 0.00035 ets variant 7 212613_at BTN3A2 6.8 5.9 6.9 0.00035 butyrophilin, subfamily 3, member A2 202942_at ETFB 0.239 0.256 0.289 0.00036 electron-transfer-flavoprotein, beta polypeptide

220960_x_at RPL22 0.538 0.624 0.488 0.00036 ribosomal protein L22 222592_s_at ACSL5 15.1 32.2 28.4 0.00036 acyl-CoA synthetase long-chain family member 5

226116_at --- 0.255 0.270 0.206 0.00037 --- 218613_at PSD3 0.069 0.062 0.132 0.00037 pleckstrin and Sec7 domain containing 3 206574_s_at LOC100131 0.979 0.978 0.984 0.00037 similar to protein tyrosine phosphatase type IVA, 062 /// member 3 /// protein tyrosine PTP4A3 210681_s_at USP15 3.3 5.9 4.4 0.00037 ubiquitin specific peptidase 15 202531_at IRF1 21.4 16.5 43.7 0.00037 interferon regulatory factor 1 218962_s_at TMEM168 0.362 0.387 0.406 0.00037 transmembrane protein 168 204440_at CD83 111.6 54.9 61.1 0.00037 CD83 molecule 204017_at KDELR3 0.410 0.279 0.290 0.00037 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor 3 14 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 221875_x_at HLA-F 7.7 5.2 11.5 0.00037 major histocompatibility complex, class I, F 203968_s_at CDC6 0.332 0.195 0.286 0.00037 cell division cycle 6 homolog (S. cerevisiae) 225636_at STAT2 3.0 2.9 4.6 0.00038 signal transducer and activator of transcription 2, 113kDa 209451_at TANK 2.1 2.8 2.9 0.00038 TRAF family member-associated NFKB activator

221492_s_at ATG3 1.7 2.1 1.8 0.00038 ATG3 autophagy related 3 homolog (S. cerevisiae)

209817_at PPP3CB 0.351 0.412 0.304 0.00038 protein phosphatase 3 (formerly 2B), catalytic subunit, beta isoform 203340_s_at SLC25A12 5.7 5.7 6.5 0.00038 solute carrier family 25 (mitochondrial carrier, Aralar), member 12 1570261_at --- 17.5 46.2 41.1 0.00038 --- 210514_x_at HLA-G 4.2 3.7 4.9 0.00038 major histocompatibility complex, class I, G 227009_at --- 0.248 0.166 0.245 0.00038 --- 218810_at ZC3H12A 10.7 18.6 19.5 0.00038 zinc finger CCCH-type containing 12A 211012_s_at LOC161527 9.2 16.8 15.6 0.00038 hypothetical protein LOC161527 /// /// PML promyelocytic leukemia 223106_at TMEM14C 0.696 0.727 0.695 0.00039 transmembrane protein 14C 216252_x_at FAS 4.0 6.6 8.6 0.00039 Fas (TNF receptor superfamily, member 6) 204994_at MX2 246.3 59.5 369.6 0.00039 myxovirus (influenza virus) resistance 2 (mouse)

235459_at --- 0.225 0.194 0.155 0.00039 --- 34689_at ATRIP /// 9.1 15.1 30.9 0.00039 ATR interacting protein /// three prime repair TREX1 exonuclease 1 201413_at HSD17B4 0.359 0.378 0.315 0.00039 hydroxysteroid (17-beta) dehydrogenase 4 213474_at KCTD7 0.281 0.329 0.193 0.00039 potassium channel tetramerisation domain containing 7 238629_x_at --- 99.2 40.5 152.8 0.00039 --- 235116_at TRAF1 29.2 12.5 18.8 0.0004 TNF receptor-associated factor 1 226401_at PARP10 3.5 6.9 6.0 0.0004 poly (ADP-ribose) polymerase family, member 10

219582_at OGFRL1 2.6 3.1 3.8 0.0004 opioid growth factor receptor-like 1 238725_at IRF1 27.0 24.8 82.4 0.0004 interferon regulatory factor 1 239461_at GALNTL2 0.117 0.203 0.129 0.0004 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase

240469_at --- 1.0 1.0 1.0 0.0004 --- 219100_at OBFC1 0.404 0.411 0.509 0.0004 oligonucleotide/oligosaccharide-binding fold containing 1 202869_at OAS1 327.5 186.8 1317.6 0.00041 2',5'-oligoadenylate synthetase 1, 40/46kDa 236018_at --- 0.685 0.642 0.692 0.00041 --- 204521_at C12orf24 0.185 0.182 0.196 0.00041 chromosome 12 open reading frame 24 202637_s_at ICAM1 996.8 275.5 159.5 0.00041 intercellular adhesion molecule 1 1553976_a_at RP11- 0.275 0.323 0.428 0.00041 deleted in a mouse model of primary ciliary 529I10.4 dyskinesia 15 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 223551_at PKIB 4.1 7.7 4.9 0.00042 protein kinase (cAMP-dependent, catalytic) inhibitor beta 210312_s_at IFT20 0.472 0.535 0.449 0.00042 intraflagellar transport 20 homolog (Chlamydomonas) 205476_at CCL20 515.5 169.7 1087.6 0.00042 chemokine (C-C motif) ligand 20 204388_s_at MAOA 0.062 0.133 0.090 0.00042 monoamine oxidase A 201517_at NCBP2 0.547 0.494 0.534 0.00042 nuclear cap binding protein subunit 2, 20kDa

202313_at PPP2R2A 2.1 2.5 2.9 0.00042 protein phosphatase 2 (formerly 2A), regulatory subunit B, alpha isoform 201350_at FLOT2 1.7 1.5 1.9 0.00042 flotillin 2 227319_at --- 0.211 0.223 0.256 0.00042 --- 226795_at --- 3.1 2.9 4.5 0.00042 --- 235279_at --- 0.135 0.142 0.078 0.00043 --- 221485_at B4GALT5 4.4 3.5 4.5 0.00043 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 5 225633_at DPY19L3 0.142 0.148 0.112 0.00043 dpy-19-like 3 (C. elegans) 230098_at PHF20L1 0.282 0.392 0.390 0.00043 PHD finger protein 20-like 1 212288_at FNBP1 0.353 0.304 0.207 0.00043 formin binding protein 1 217719_at EIF3L 0.348 0.341 0.253 0.00043 eukaryotic translation initiation factor 3, subunit L

200006_at PARK7 0.592 0.708 0.592 0.00043 Parkinson disease (autosomal recessive, early onset) 7 202509_s_at TNFAIP2 6.2 5.4 8.9 0.00044 tumor necrosis factor, alpha-induced protein 2

235742_at RHOC 7.2 6.0 12.5 0.00044 Full length insert cDNA clone YP95A10 212037_at PNN 0.467 0.378 0.411 0.00044 pinin, desmosome associated protein 218633_x_at ABHD10 0.267 0.318 0.270 0.00044 abhydrolase domain containing 10 214042_s_at RPL22 0.309 0.392 0.238 0.00044 ribosomal protein L22 207850_at CXCL3 21.3 55.3 20.6 0.00044 chemokine (C-X-C motif) ligand 3 1553959_a_at B3GALT6 2.5 2.0 2.4 0.00044 UDP-Gal:betaGal beta 1,3-galactosyltransferase polypeptide 6 243222_at --- 4.2 5.1 5.4 0.00044 --- 201743_at CD14 11.8 30.7 19.5 0.00044 CD14 molecule 202734_at TRIP10 2.1 1.8 1.9 0.00044 thyroid hormone receptor interactor 10 200814_at PSME1 3.7 3.7 5.4 0.00044 proteasome (prosome, macropain) activator subunit 1 (PA28 alpha) 207196_s_at TNIP1 8.1 15.9 16.7 0.00044 TNFAIP3 interacting protein 1 1552378_s_at RDH10 0.083 0.103 0.037 0.00044 retinol dehydrogenase 10 (all-trans) 214838_at SFT2D2 12.0 7.8 11.4 0.00045 SFT2 domain containing 2 226711_at FOXN2 2.1 3.0 2.7 0.00045 forkhead box N2 222068_s_at LRRC50 28.2 15.9 23.8 0.00045 leucine rich repeat containing 50 209694_at PTS 0.324 0.354 0.360 0.00045 6-pyruvoyltetrahydropterin synthase 224606_at KLF6 3.5 2.8 4.1 0.00045 DNA-binding protein CPBP (CPBP) 218124_at RETSAT 0.663 0.680 0.579 0.00046 retinol saturase (all-trans-retinol 13,14-reductase)

204924_at TLR2 46.6 17.2 79.8 0.00046 toll-like receptor 2 16 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 235812_at TMEM188 1.7 1.8 1.5 0.00046 transmembrane protein 188 234523_at --- 1.0 1.0 1.0 0.00046 --- 225394_s_at ZCRB1 0.614 0.539 0.604 0.00046 zinc finger CCHC-type and RNA binding motif 1

223502_s_at TNFSF13B 243.5 582.7 187.6 0.00046 tumor necrosis factor (ligand) superfamily, member 13b 225850_at SFT2D1 1.6 1.6 1.6 0.00046 SFT2 domain containing 1 219685_at TMEM35 0.051 0.037 0.059 0.00046 transmembrane protein 35 224973_at FAM46A 4.0 4.7 7.3 0.00046 family with sequence similarity 46, member A

208973_at ERI3 0.475 0.573 0.493 0.00046 exoribonuclease 3 207388_s_at PTGES 20.0 25.8 56.4 0.00046 prostaglandin E synthase 240088_at PDE5A 0.179 0.177 0.186 0.00046 phosphodiesterase 5A, cGMP-specific 202510_s_at TNFAIP2 41.1 31.5 149.4 0.00047 tumor necrosis factor, alpha-induced protein 2

1553286_at ZNF555 0.406 0.379 0.532 0.00047 zinc finger protein 555 823_at CX3CL1 65.5 24.5 111.6 0.00047 chemokine (C-X3-C motif) ligand 1 233632_s_at XRN1 2.2 3.2 3.2 0.00047 5'-3' exoribonuclease 1 229958_at CLN8 0.093 0.189 0.175 0.00047 ceroid-lipofuscinosis, neuronal 8 (epilepsy, progressive with mental retardation 208768_x_at RPL22 0.465 0.546 0.388 0.00047 ribosomal protein L22 201227_s_at NDUFB8 0.489 0.445 0.530 0.00047 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 8, 19kDa 1562966_at KIAA1217 8.9 7.0 6.2 0.00047 KIAA1217 201641_at BST2 276.1 314.8 1074.3 0.00047 bone marrow stromal cell antigen 2 1558250_s_at --- 0.162 0.212 0.313 0.00047 --- 1553713_a_at RHEBL1 9.3 5.4 6.7 0.00047 Ras homolog enriched in brain like 1 200904_at HLA-E 5.3 5.5 8.5 0.00047 major histocompatibility complex, class I, E 205240_at GPSM2 0.461 0.425 0.326 0.00047 G-protein signaling modulator 2 (AGS3-like, C. elegans) 220363_s_at ELMO2 3.8 5.6 6.9 0.00048 engulfment and cell motility 2 217771_at GOLM1 13.6 12.9 20.7 0.00048 golgi membrane protein 1 202432_at PPP3CB 0.400 0.356 0.523 0.00048 protein phosphatase 3 (formerly 2B), catalytic subunit, beta isoform 235042_at XIRP1 84.9 25.0 75.6 0.00048 xin actin-binding repeat containing 1 203803_at PCYOX1 0.199 0.260 0.203 0.00048 prenylcysteine oxidase 1 205205_at RELB 25.9 20.4 23.0 0.00049 v-rel reticuloendotheliosis viral oncogene homolog B 218421_at CERK 0.112 0.132 0.249 0.00049 ceramide kinase 225076_s_at ZNFX1 5.1 8.3 9.1 0.00049 zinc finger, NFX1-type containing 1 202961_s_at ATP5J2 0.731 0.804 0.742 0.00049 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F2 231959_at LIN52 4.3 6.0 9.4 0.00049 lin-52 homolog (C. elegans) 204589_at NUAK1 0.115 0.115 0.115 0.00049 NUAK family, SNF1-like kinase, 1 209390_at TSC1 0.431 0.382 0.470 0.00049 tuberous sclerosis 1 215646_s_at VCAN 0.114 0.105 0.092 0.0005 versican 202863_at SP100 3.1 4.1 5.2 0.0005 SP100 nuclear antigen 17 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 220731_s_at NECAP2 2.3 2.4 2.3 0.0005 NECAP endocytosis associated 2 223268_at C11orf54 0.459 0.398 0.482 0.0005 chromosome 11 open reading frame 54 1558115_at RECK 0.687 0.607 0.656 0.0005 reversion-inducing-cysteine-rich protein with kazal motifs 224232_s_at PRELID1 2.1 2.4 2.0 0.0005 PRELI domain containing 1 215078_at SOD2 6.6 17.3 10.6 0.0005 superoxide dismutase 2, mitochondrial 221021_s_at CTNNBL1 5.8 4.8 10.1 0.0005 catenin, beta like 1 226336_at PPIA 0.297 0.236 0.336 0.0005 peptidylprolyl isomerase A (cyclophilin A) 207740_s_at NUP62 4.1 6.4 3.6 0.00051 nucleoporin 62kDa 207761_s_at METTL7A 0.139 0.109 0.187 0.00051 methyltransferase like 7A 230893_at DNAJC21 0.155 0.272 0.206 0.00051 DnaJ (Hsp40) homolog, subfamily C, member 21

227092_at --- 0.196 0.320 0.329 0.00051 --- 230499_at --- 12.0 18.7 36.1 0.00051 --- 214508_x_at CREM 9.3 5.1 8.5 0.00051 cAMP responsive element modulator 213191_at TICAM1 4.1 3.7 3.3 0.00051 toll-like receptor adaptor molecule 1 238601_at PHKB 0.216 0.238 0.176 0.00051 phosphorylase kinase, beta 230266_at RAB7B 0.088 0.157 0.080 0.00051 RAB7B, member RAS oncogene family 216466_at NAV3 4.7 3.2 3.8 0.00051 neuron navigator 3 224994_at CAMK2D 2.0 2.0 2.1 0.00052 calcium/calmodulin-dependent protein kinase II delta 203045_at NINJ1 18.9 12.4 41.2 0.00052 ninjurin 1 223443_s_at FLJ32065 0.220 0.342 0.353 0.00052 hypothetical protein FLJ32065 229441_at PRSS23 0.065 0.163 0.061 0.00052 CDNA PSEC0048 fis, clone NT2RP2000028, highly similar to Serine protease 23 prec 204088_at P2RX4 3.8 3.1 2.5 0.00053 purinergic receptor P2X, ligand-gated ion channel, 4 201486_at RCN2 0.412 0.421 0.422 0.00053 reticulocalbin 2, EF-hand calcium binding domain

226748_at LYSMD2 2.7 4.4 3.7 0.00053 LysM, putative peptidoglycan-binding, domain containing 2 221766_s_at FAM46A 5.0 4.7 8.2 0.00053 family with sequence similarity 46, member A

206790_s_at NDUFB1 0.692 0.743 0.733 0.00053 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 1, 7kDa 237287_at WDR34 0.904 0.878 0.858 0.00053 CDNA FLJ57659 complete cds, highly similar to WD repeat protein 34 202365_at UNC119B 0.185 0.124 0.154 0.00054 unc-119 homolog B (C. elegans) 228955_at --- 0.173 0.188 0.196 0.00054 --- 226793_at LOC283267 0.470 0.430 0.460 0.00054 hypothetical LOC283267

226282_at --- 0.171 0.099 0.120 0.00054 --- 233748_x_at PRKAG2 0.412 0.349 0.485 0.00054 protein kinase, AMP-activated, gamma 2 non- catalytic subunit 236253_at --- 1.0 1.0 1.0 0.00054 --- 201540_at FHL1 0.114 0.170 0.085 0.00054 four and a half LIM domains 1 209476_at TXNDC1 3.9 4.7 4.5 0.00054 thioredoxin domain containing 1 18 Fold Ratio (Stim/Con) Probeset ID Symbol FS087 MRC5 N78 p-value Gene Title 224116_at --- 1.0 1.0 1.1 0.00054 --- 239050_s_at --- 0.349 0.312 0.452 0.00054 --- 204958_at PLK3 2.4 3.5 2.6 0.00055 polo-like kinase 3 (Drosophila) 222064_s_at AARSD1 0.466 0.455 0.511 0.00055 alanyl-tRNA synthetase domain containing 1

213038_at RNF19B 10.8 28.7 16.1 0.00055 ring finger protein 19B 212498_at --- 0.358 0.352 0.470 0.00055 --- 226161_at SLC30A6 0.601 0.710 0.610 0.00055 solute carrier family 30 (zinc transporter), member 6