Inhibition of Protein Disulfide Isomerase Induces Differentiation of Acute Myeloid Leukemia Cells

Acute Myeloid Leukemia SUPPLEMENTARY APPENDIX Inhibition of protein disulfide isomerase induces differentiation of acute myeloid leukemia cells

Justyna Chlebowska-Tuz, 1,2,3 Olga Sokolowska, 1,2,4 Pawel Gaj, 1,5 Michal Lazniewski, 6,7 Malgorzata Firczuk, 1 Karolina Borowiec, 1 Hanna Sas-Nowosielska, 8 Malgorzata Bajor, 1 Agata Malinowska, 9 Angelika Muchowicz, 1 Kavita Ramji, 1 Piotr Stawinski, 10 Mateusz Sobczak, 2 Zofia Pilch, 1 Anna Rodziewicz-Lurzynska, 11 Malgorzata Zajac, 12 Krzysztof Giannopoulos, 12 Przemyslaw Juszczynski, 13 Grzegorz W. Basak, 14 Dariusz Plewczynski, 6,15 Rafal Ploski, 10 Jakub Golab 1,16 and Dominika Nowis 1,2,17

1Department of Immunology, Medical University of Warsaw; 2Laboratory of Experimental Medicine, Center of New Technologies, Univer - sity of Warsaw; 3Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw; 4Postgraduate School of Molecular Medicine, Medical University of Warsaw; 5Laboratory of Human Cancer Genetics, Center of New Technologies, University of Warsaw; 6Laboratory of Functional and Structural Genomics, Center of New Technologies, University of Warsaw; 7Department of Physical Chem - istry, Faculty of Pharmacy, Medical University of Warsaw; 8Laboratory of Imaging Tissue Structure and Function, Nencki Institute of Ex - perimental Biology, Polish Academy of Sciences, Warsaw; 9Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw; 10 Department of Medical Genetics, Center of Biostructure Research, Medical University of War - saw; 11 Department of Laboratory Diagnostics, Faculty of Health Sciences, Medical University of Warsaw; 12 Department of Experimental Hematooncology, Medical University of Lublin; 13 Department of Experimental Hematology, Institute of Hematology and Transfusion Medicine, Warsaw; 14 Department of Hematology, Oncology and Internal Diseases, Medical University of Warsaw; 15 Faculty of Mathe - matics and Information Science, Warsaw University of Technology, Warsaw; 16 Center for Preclinical Research and Technology, Medical University of Warsaw and 17 Genomic Medicine, Medical University of Warsaw, Poland

Received: February 3, 2018. Accepted: July 10, 2018. Pre-published: July 12, 2018. Correspondence: [email protected] or [email protected] Inhibition of protein disulfide isomerase induces differentiation of AML cells

Supplementary data

Supplementary Materials and Methods

SK053 and its analogues. Chemical structures of SK053 and its analogs (Supplementary Fig. S6A-C) were created using Marvin Sketch software ver. 15.9.28, Chem Axon Ltd, Cambridge, MA, USA. Details of chemical synthesis of SK053 are described elsewhere.1

AML cell lines. KG1 (ATCC-CCL-246), HL-60 (ATCC-CCL-240), HeLa (ATCC-CCL-2) and HS-5 (ATCC-CRL-11882) were purchased from American Type Culture Collection (ATCC, LGC Standards, Teddington, UK). NB4 (ACC 207) cell line was purchased from German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). MOLM14 cell line was kindly provided by Prof. Przemyslaw Juszczynski (Department of Experimental Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland).

AML cell lines culture conditions. NB4, MOLM14, HS-5 and HL-60 cells were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone), 100 µg/ml streptomycin, 100 U/ml penicillin (Sigma-Aldrich). KG1 cells were cultured in IMDM (Gibco) supplemented with 20% heat-inactivated fetal bovine serum (Hyclone) and antibiotics as stated above. HeLa cells were cultured in DMEM (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone) and antibiotics as stated above. All cells were cultured at 37°C in a fully humidified atmosphere of 5% CO2. In all experiments, cells were incubated with SK053, all-trans retinoic acid (ATRA; Sigma- Aldrich) or phorbol esters (PMA; Sigma-Aldrich) as positive control for differentiation and 0.1% dimethyl sulfoxide (DMSO; Sigma-Aldrich) as solvent control. Medium with drugs was replaced every third day of incubation time. Compounds stocked dissolved in DMSO were stored at –20°C until used.

Cell viability and growth assessment. For trypan blue exclusion assay cells were plated in triplicates at 2×105 cells per well in 12-well culture plates in 1 ml of culture medium with indicated SK053 concentrations. Cell numbers and viability were estimated with trypan blue (Sigma-Aldrich) exclusion assay, by counting cells from each well in duplicates in Bürker chamber (Heinz Herenz Medizinalbedarf, Hamburg, Germany). Percentage of dead cells was also evaluated in flow cytometry with propidium iodide (PI; 2 µg/ml, Sigma-Aldrich) using LSR II Fortessa cytometer (BD Biosciences, San Jose, CA, USA) and BD FACSDiva software version 8 (BD Biosciences). For trypan blue exclusion assay (Fig. 4F, Supplementary Fig. S6C,D) cells were plated in duplicates at 2×105 cells per well in 12-well culture plates in 1 ml of culture medium with indicated SK053 concentrations. Cell numbers and viability were estimated with trypan blue (Bio-Rad) exclusion assay, by counting cells from each well in duplicates in TC20 Automated Cell Counter (Bio-Rad).

AML differentiation assays. For differentiation experiments, cells were seeded at a density of 2×105 cells/ml in 6-well plate and treated with indicated concentrations of SK053. In some experiments 1 μM ATRA, 5 μM ATRA or 50 ng/ml PMA served as positive differentiation controls for APL (HL-60, NB4), MOLM14 and KG1 cell lines, respectively. To examine cellular morphology, cells were cytospun using Cytospin4 centrifuge (Thermo Fisher Scientific) at 100×g for 10 min at RT, stained with May Grünwald-Giemsa (MGG; Merck KGaA, Darmstadt, Germany) and observed using Eclipse 80i (Nikon) microscope equipped with QICAM Fast 1394 camera (QImaging, Surrey, BC, Canada and Image Pro Plus

1 software version 7.0 (Media Cybernetics, Rockville, MD, USA). Myeloid differentiation was assessed by surface expression of CD11b. Briefly, 3×105 cells were collected, washed with PBS and incubated for 30 min at RT with anti-human anti-CD11b-Alexa Fluor488 antibodies, cat. no. 11-0118-42 (eBioscience, Thermo Fisher Scientific) diluted in 2% bovine serum albumin (BSA; Sigma-Aldrich) followed by PBS wash and viability staining with 7AAD (eBioscience) according to manufacturer’s protocol. Fluorescence intensity was evaluated using FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo software version 10.2 (FlowJo LLC). Conventional semi-quantitative microscopic nitroblue tetrazolium (NBT; Sigma-Aldrich) assay was used to determine the production of superoxide anion upon stimulation with PMA in differentiated AML cells. To this end, 1×106 of cells were collected, washed with PBS and suspended in 100 μl RPMI 1640 and mixed with an equal volume of 2 mg/ml NBT dissolved in PBS containing 2 μg/ml PMA and incubated at 37°C for 2 h. Microscopic observation revealed cells containing blue NBT formazan deposits in MGG stained slides. Moreover, modified quantitative NBT assay was established and implemented by dissolving the blue formazan particles from equal cell numbers in 100 μL of 2M sodium hydroxide, followed by measurement of absorbance at 620 nm using ASYS UVM 340 microplate reader (Biochrom, Cambridge, UK).

HS5 and HL-60 co-culture. Cytostatic/cytotoxic effects of SK053 on HL-60 cells in coculture with stroma cell line HS5 was performed using a transwell system (Falcon; cat. No. 734- 0038, Thermo Fisher Scientific). HS5 cells were seeded at a density 5×104 /well on 24-well plate in 0.5 ml medium into the lower compartment of transwell. Next, 3×104 of HL-60 cells were seeded into the upper compartment. Medium with SK053 was changed every day. Upon 72 h incubation with SK053, the cytostatic/cytotoxic effects were assessed using propidium iodide staining (final concentration 2.5 μg/ml; Sigma-Aldrich). Red fluorescence was analyzed in LSR Fortessa flow cytometer (BD Biosciences) and FlowJo software version 10.2 (FlowJo LLC, Ashland, OR, USA) and BD FACSDiva software version 8 (BD Biosciences).

LICs assessment. Surface expression of CD34, CD38 and CD123 markers was used to evaluate the percentage of leukemia initiating cells (LICs) in KG1 cell line incubated for the indicated times with SK053. For the analysis 5×105 cells were collected, washed with PBS and incubated for 30 min at RT with anti-human mAbs - anti-CD34-APC, cat. no. 555824; anti-CD38-V450, cat. no. 646851, anti-CD123-PerCP-Cy5.5, cat. no. 558714 (all obtained from BD Biosciences, San Jose, CA, USA) diluted according to manufacturer’s protocol in 2% BSA. Cells were washed with PBS and flow cytometry analysis was performed on the LSR II Fortessa cytometer (BD Biosciences) with BD FACSDiva software version 8 (BD Biosciences) or FlowJo software version 10.2 (FlowJo LLC).

Colony formation assay. To determine the clonogenic potential, KG1 cells were pre-treated for 72h with SK053, washed with PBS and then 5×103 cells were plated in triplicates in cytokine-enriched methylcellulose (MethoCult™Optimum Without EPO, Cat. No H4035, StemCell Technologies, Inc.) on Smart Dishes (Cat. No 27371 StemCell Technologies, Inc). Colonies containing more than 20 cells were counted after 14 days of culture. Images of colonies were acquired using Olympus CKX52 microscope and analyzed with ImageJ software (https://imagej.nih.gov/).

Incubation of recombinant proteins with SK053. For mass spectrometry and binding assays huPDI protein was reduced by 15-min incubation with 10 mM DTT at 37°C followed by a dialysis to 10 mM Tris pH7.5 and 30-min incubation with a 10× molar excess of SK053 at 37°C. To oxidize –SH groups of PDI for binding assays, the protein was left in the fridge for 24 or 48 h. Next, 10 µg of reduced or oxidized huPDI was incubated for 30 min at 37°C with a 10× molar excess of SK-BIO. 200 ng of each protein was separated with SDS-PAGE followed by Western blotting using anti-biotin monoclonal antibody (Supplementary Tab. S9). Gels stained with Coomasie blue (Bio-Rad, Hercules, CA, USA) served as loading controls.

PDIA1-d4 reduced with 5mM DTT was incubated with SK053 in 1:10 molar ratio for 30 min at 37°C in 10 mM Tris buffer, pH 7.5. The binding of SK053 to PDIA1-d4 was assessed by mass spectrometry using LTQ Orbitrap Velos mass spectrometer (ThermoElectron).

Gene silencing using ready to use lentivirus-expressed shRNAs. Ready to use MISSION pLKO.1-puro shRNA lentivirus particles targeting TXN [TXN(a)], PDI [PDI(a), PDI(b)] as well as non-targeting shRNA scramble control (NTC) (Supplementary Tab. S10) were purchased from Sigma-Aldrich. Transduction was performed according to the manufacturer's instructions. At 72 h post transduction successfully infected HL-60 cells were selected with 2 μg/ml puromycin (Sigma-Aldrich), knock-down was validated by Western blotting 5 days post transduction. PDI(b) hairpin did not silence P4HB expression.

Gene silencing using custom packaged lentivirus-expressed shRNAs. Lentiviruses for shRNA were produced by co-transfecting (calcium chloride) HEK293T cells with MISSION pLKO.1-puro shRNA plasmids targeting TXN [TXN(b), TXN(c), TXN(d)], PDI [PDI(c), PDI(d), PDI (e)], CEBPA [CEBPA(a), CEBPA(b), CEBPA (c)] as well as non-targeting shRNA scramble control (NTC) (Supplementary Tab. S10) (Sigma-Aldrich) together with packaging plasmids pMD2.G and psPAX2 (pMD2.G and psPAX2 plasmids were generous gifts from prof. Didier Trono (Ecole polytechnique federale de Lausanne, Switzerland).Transduction was performed according to the manufacturer's instructions. At 72 h post transduction successfully infected cells were selected with 2 μg/ml puromycin (Sigma-Aldrich), knock- down was validated by Western blotting 7 days post transduction. TXN(d), CEBPA(b) and PDI(c) hairpins did not silence expression of the corresponding genes. PDI(e) hairpin showed off target effects (silenced ERO1α expression).

RNA extraction and quantitative Real-Time PCR RNA concentration was measured with NanoDrop spectrophotometer (Thermo Fisher Scientific). A total of 0.5 μg RNA was used for cDNA synthesis primed with oligo(dT) using Native AMV Reverse Transcriptase (EurX, Gdansk, Poland) in a final volume of 10 μl. For each qPCR run, the samples were measured in duplicates to estimate their reproducibility. Primers sequences and probes numbers are shown in Supplementary Tab. S8. Samples in which the cDNA was omitted were used as negative controls. The results were analyzed after 40 cycles of amplification using LightCycler 480 Software 1.5 and normalized for the content of the housekeeping genes (β2-microglobulin and ribosomal protein L29). The fold change for each gene was calculated using a user-non-influent, second derivative method. The specificity of the reactions was confirmed by analysis of the PCR product in 2% agarose gel.

Immunoprecipitation. HL-60 cells (5×106) were incubated for 4 h in culture media without FBS containing 100 μM SK-BIO or SK-IN. Next, the cells were lysed in 0.1% (v/v) NP-40 in PBS supplemented with Complete protease inhibitors cocktail (Roche). Equal amounts of proteins were incubated with 5 µg of anti-tioredoxin (TXN) (Abcam, Cambridge, UK; clone 3A1) antibody per 500 ml of lysate, 5 µg of anti-PDI (Abcam; clone RL90) antibody per 500 ml of lysate or without antibodies for 2 h at room temperature (RT) with gentle agitation. Next, protein G-coated beads (Pierce, Thermo Fisher Scientific) were added to the lysates for the subsequent 16 h incubation at 4°C with gentle agitation. After extensive washes with lysis buffer, beads-bound proteins were eluted by boiling in SDS sample buffer. Then, 20 µL of samples were electrophoresed and probed with anti-TXN, anti-PDI, and anti-biotin antibodies using working concentrations as described in Supplementary Tab. S9.

PDI enzymatic assay. PDI enzymatic activity was assessed in a turbidimetric assay measuring the thiol isomerase-catalyzed reduction of insulin in the presence of DTT. The absorption of aggregated reduced insulin chains was measured at 650 nm using Asys UV340M plate reader (Biochrom). The enzymatic activity assay mixture was prepared in the final volume of 150 μl. Briefly, 1.7 μM recombinant PDI protein was preincubated for 1h at

37°C with indicated SK053 concentrations in the presence of 2 mM EDTA in 100 mM potassium phosphate buffer, pH 7.4. Next, 135 μL of 270 μM DTT (Thermo Fisher Scientific) and 545 μM of bovine insulin (Sigma-Aldrich) mixture dissolved in reaction buffer was added. The plate was incubated at 25°C and absorbance was read every 5 min for subsequent 60 min. IC50 was determined using SigmaPlot software (Systat Software Inc., San Jose, CA, USA).

Fluorescence lifetime measurements by time-correlated single-photon counting (TCSPC) The day before transfection HeLa cells were plated 2.5×105 cells per well on 35 mm glass bottom dishes (Cell E&G, San Diego, CA, USA) covered with poly-L-lysine (Sigma-Aldrich) and then cells were transfected with 2 µg of roGFPiE containing plasmid 2 (kindly provided by Professor David Ron from University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Cambridge, UK) using 5 µL Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific), according to the manufacturer’s protocol. Next day, cells were incubated with DMSO (solvent control) for 8h, 25 µM SK053 for 8h or 100 µM DTT for 30 min. Fluorescence lifetime imaging (FLIM) data were collected using SP8 confocal microscope equipped with TCSPC module (PicoHarp300, PicoQuant, Berlin, Germany). Moreover the system was equipped with an incubation chamber suitable to maintain the live cells and optics at 37°C, 63× water immersion NA 1.2 objective lens and with confocal pinhole set to 2 Airy units. To excite GFP fluorescence white light laser with wavelength set to 488 nm and 40 kHz repetition rate was used. GFP fluorescence was detected in 500-550 nm band with APD detector. FLIM data were collected in TCSPC mode using PicoHarp300 (PicoQuant). To avoid photo toxicity and minimize perturbations due to cells motility, FLIM images were collected 64×64 pixel format, with pixel size set to 14 μm. FLIM data were collected until 5000 counts in the brightest pixel of an image were acquired. Data analysis and fitting were performed using FLIMfit software (Imperial College London, UK).

Western blot. HL-60 cells were washed twice with ice-cold PBS, lysed in RIPA buffer (50 mmol/L Tris base, 150 mmol/L NaCl, 1% NP-40, 0.25% sodium deoxycholate, and 1 mmol/L EDTA) or cytoplasmic/nuclear fractions of proteins were extracted using NE-PER Kit (Pierce). Lysis buffers were supplemented with Complete protease inhibitor cocktail (Roche). The lysates were then cleared by centrifugation at 15,000×g for 10 min. Protein concentration in supernatants was evaluated with Bio-Rad Protein Assay (Bio-Rad). Next, samples were boiled for 5 min in 5× Laemmli Sample Buffer. Equal amounts of lysates were subjected to SDS-PAGE, transferred onto a nitrocellulose membrane (Schleicher and Schuell, Sigma-Aldrich) and then blocked with Tris-buffered saline (pH 7.4) and 0.05% Tween 20 supplemented with 5% BSA. Next, membranes were probed with primary (listed in Supplementary Tab. S9) and corresponding HRP-coupled secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) before visualization using self-made chemiluminescence reagent (100 mM Tris pH 8.0, 1.25 mM luminol, 0.2 mM coumaric acid, 0.006% hydrogen peroxide) with Stella 8300 bioimager (Raytest, Angleur, Belgium).

RNA-seq libraries preparation. A total of 1 µg RNA was treated with Turbo-DNase (Life Technologies) according to the manufacturer’s instructions. Then, rRNA was removed from samples using a Ribo-Zero Kit (Epicentre) according to the manufacturer’s protocol, and samples were spiked-in with external RNA (ERCC RNA Spike-In Mix; Life Technologies). RNA libraries were prepared using KAPA Stranded RNA-Seq Library Preparation Kit (Kapa Biosystems) according to the manufacturer’s instructions. Quality of libraries was determined by chip electrophoresis performed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.).

RNA-seq data acquisition and analysis. Ribo-depleted total RNA isolated from HL-60 cell line incubated with SK053 in duplicates were used to prepare strand-specific libraries (dUTP

RNA). These libraries were subsequently sequenced using an Illumina HiSeq sequencing platform to the average number of 21.9 million reads per sample in 100-nt pair-end mode. The quality control of obtained sequences was performed using FastQC software. Reads were than mapped to the reference human genome (hg19) using STAR short-read aligner yielding an average of 76.4% uniquely mapped reads. Read processing, filtering and counting were performed using HTseq software. Differential expression analyses were performed using the EdgeR following gene filtration and raw read count normalization against differences in library sizes using TMM algorithm. The Gene Ontology (GO) (BP: Biological Process or MF: Molecular Function) enrichment analysis was carried out in Cytoscape (BiNGO) and topGO using the lists of differentially expressed genes (FDR<0.01) between cells incubated with SK053 and the control cells (DMSO). The background set of genes used in the GO analysis consisted of the filtered set of genes previously used in the RNAseq DGE analysis. The results were adjusted for multiple hypothesis testing using Benjamini-Hochberg FDR method.

Expression of human PDI. cDNA encoding human PDI was cloned and expressed in a prokaryotic protein expression system followed by nickel affinity chromatography and subsequent gel filtration as described.1

Cloning, expression, purification of d4 domain (4th thioredoxin-like domain) of huPDIA1. The d4 domain of human PDIA1 protein, corresponding to the sequence encoding amino acids 368-475, was amplified by PCR using the following primers: FPDIA1d4: 5' GGCGAATTCCCTGTCAAGGTGCTTGTTGG and RPDIA1d4: 5' GGCCTCGAGTTATTGGCCACCGCTCTCTAGG. cDNA from human prostate cancer cell line DU-145 (ATCC No HTB-81) was used as a template. The PCR product was cloned into EcoRI and XhoI recognition sites of pET15-mod plasmid, a derivative of Novagen pET15b(+) vector, containing 6 N-terminal histidine codons (a generous gift from Prof. Mattias Bochtler from the International Institute of Molecular and Cell Biology, Warsaw, Poland). The sequence of the insert was confirmed by Sanger sequencing. For protein expression, the plasmid pET15-mod-PDIA1-d4 was transformed to E. coli Rosetta strain. Cells were grown at 37°C until reached OD600 = 0.6-0.7, at which point 1 mM isopropyl β-D-1- thiogalactopyranoside (IPTG) was added and the cultures were shifted to 30°C for additional 20 h. Recombinant protein was purified by nickel affinity and gel filtration chromatography as described.3

Pulldown of SK-BIO binding proteins and mass spectrometry. HL-60 cells (30×106) were incubated for 4h in culture media without FBS containing 100 μM of biotinylated enzymatically active analogs of SK053 (referred to as SK-BIO, Supplementary Fig. S1B) or an inactive analog lacking electrophile group attacking the nucleophilic cysteine residues (referred to as SK-IN, Fig. S1C). After cell lysis and extensive washes, beads-bound proteins were eluted with 5 mM biotin (Sigma-Aldrich), 5 mM biotin and 5 mM urea (Avantor Performance Materials, Gliwice, Poland) or by boiling in SDS sample buffer. Band detected in 12% polyacrylamide silver stained gel in SK-BIO sample was excised and analyzed by mass spectrometry using LTQ Orbitrap Velos mass spectrometer (ThermoElectron, San Jose, CA, USA). The obtained reads were compared with NCBI, UniProt database and analyzed using Mascot software.

Mass spectrometry of huPDI incubated with SK053. Following incubation with 10 x molar excess of SK053, 30 µg of recombinant huPDI was digested overnight with 10 ng/µL trypsin (Promega, Madison, WI, USA), then reduced with 100 mM DTT for 30 min at 56°C, and alkylated with 50 mM iodoacetamide in the dark for 45 min at 24°C. The resulting peptide mixtures were applied to an RP-18 precolumn (Waters, Milford, MA, USA) using water that contained 0.1% formic acid as a mobile phase and then transferred to the RP-18 column (75 µM internal diameter; Waters) of the nanoACQUITY UPLC system (Waters) using an ACN gradient (0–30% ACN in 45 min) in the presence of 0.1% formic acid at a flow rate of 250

5 nL/min. The column outlet was coupled directly to the ion source of an LTQ Orbitrap Velos mass spectrometer (ThermoElectron, San Jose, CA, USA) working in the regime of data- dependent MS to MS/MS switch. Two blank runs that ensured the absence of cross- contamination from previous samples preceded each analysis. The obtained mass spectra were preprocessed with MascotDistiller software (v. 2.2.1, Matrix Science, Manchester, UK). The search parameters were set to the following: enzyme (semiTrypsin), variable modifications (cysteine carbamidomethylation, methionine oxidation, and SK053 modification) and the mass shift (282.157957 Da) for cysteine modification was calculated.

Molecular Dynamics simulation. To perform ensemble docking, a set of conformations of the reduced form of human PDI (pdb|4ekz) was generated using molecular dynamics simulation. The MD simulation was performed with the OpenMM program4 using the AMBER99sb force field and TIP3P water molecules (explicit solvent) with periodic boundary conditions. The thickness of the water box was 0.8 nm. Non-bond interactions were truncated at 1 nm. The simulation consisted of 3 stages: a 40 ps cooling to 0°K, a 100 ps warming to 300°K and a 20-ns production run. The unrestrained production run was carried out at a constant temperature (300°K) using the Langevin thermostat with a collision frequency of 1 ps-1 and at a constant pressure (1 atm) using a MonteCarlo barostat. The trajectories during production run were saved every 10 ps generating a total 2,000 conformations.

Conformation selection. Out of 2000 conformations obtained with MD simulation 90 were chosen for further analysis with ensemble molecular docking. Three criteria were chosen, each independently selecting 30 conformations. First, the best 30 conformations with the lowest total energy among those obtained in the production run were selected. Second, an RMSD matrix was calculated for all 2000 conformations and was subsequently used for clustering using an agglomerative clustering algorithm with UPGMA criterion. The clustering produced 30 clusters out of which a single medoid conformation was chosen. The final 30 conformations where selected based on their ability to represent the variability of distance observed between the sulfur atom of Cys397 and the sulfur atom of Cys400.

Covalent docking of SK053 to human PDI. In the next step, covalent docking of truncated SK053 (lacking the leaving group, Fig. S1d), to the previously selected 90 conformations was performed using the GOLD program.5 The covalent docking site was defined as a sphere of 12 Å radius centered at the Cys400 sulfur atom. Covalent docking simulations to Cys400 and Cys397 were carried out independently. Prior to docking, a pair of atoms that form a covalent bond was specified and all the water molecules present after MD simulation were removed. Otherwise, the docking studies were performed using the program’s default settings. For each GA run, a maximum of 100,000 operations was performed on a set of five groups with a population of 100 individuals. The annealing parameters were used as default: cutoff values of 3.0 Å for hydrogen bonds and 4.0 Å for van der Waals interactions. Early termination of the calculations was allowed if the RMSD of the three highest scored conformations were within 1.5 Å. The docking score between PDI and SK053 was calculated using the GoldScore energy based scoring function, which takes into account intra- and intermolecular hydrogen bonding interaction energy, van der Waals energy and ligand torsion energy.

Supplementary references

1. Muchowicz A, Firczuk M, Chlebowska J, Nowis D, Stachura J, Barankiewicz J, et al. Adenanthin targets proteins involved in the regulation of disulphide bonds. Biochem Pharmacol. 2014;89(2):210-6.

2. Avezov E, Cross BC, Kaminski Schierle GS, Winters M, Harding HP, Melo EP, et al. Lifetime imaging of a fluorescent protein sensor reveals surprising stability of ER thiol redox. J Cell Biol. 2013;201(2):337-49.

3. Klossowski S, Muchowicz A, Firczuk M, Swiech M, Redzej A, Golab J, et al. Studies toward novel peptidomimetic inhibitors of thioredoxin-thioredoxin reductase system. J Med Chem. 2012;55(1):55-67.

4. Eastman P, Friedrichs MS, Chodera JD, Radmer RJ, Bruns CM, Ku JP, et al. OpenMM 4: A Reusable, Extensible, Hardware Independent Library for High Performance Molecular Simulation. J Chem Theory Comput. 2013;9(1):461-9.

5. Jones G, Willett P, Glen RC, Leach AR, Taylor R. Development and validation of a genetic algorithm for flexible docking. J Mol Biol. 1997;267(3):727-48.

Descriptions to supplementary tables

Supplementary Table S1. Genes differentially expressed between HL-60 cells treated for 48 h with 10 µM SK053 and control cells (DMSO).

Supplementary Table S2. Results of Gene Ontology (Biological Process) analysis carried out in Cytoscape {BiNGO}c using the list of genes differentially expressed (FDR < 0.01) between HL-60 cells treated for 48 h with 10 µM SK053 and the control cells (DMSO).

Supplementary Table S3. Results of Gene Ontology (Molecular Function) analysis carried out in Cytoscape {BiNGO} using the list of genes differentially expressed (FDR < 0.01) between HL-60 cells treated for 48 h with 10 µM SK053 and the control cells (DMSO).

Supplementary Table S4. Genes differentially expressed between HL-60 cells treated for 120 h with 10 µM SK053 and control cells (DMSO).

Supplementary Table S5. Results of Gene Ontology (Biological Process) analysis carried out in Cytoscape {BiNGO} using the list of genes differentially expressed (FDR < 0.01) between HL-60 cells treated for 120 h with 10 µM SK053 and the control cells (DMSO).

Supplementary Table S6. Results of Gene Ontology (Molecular Function) analysis carried out in Cytoscape {BiNGO} using the list of genes differentially expressed (FDR < 0.01) between HL-60 cells treated for 120 h with SK053 and the control cells (DMSO).

Supplementary Table S7 - Top proteins pulled down using biotinylated SK053 from HL-60 AML cells identified in mass spectrometry gi|39794653 Keratin 1 [Homo sapiens] gi|375314779 keratin 1 [Homo sapiens] gi|435476 cytokeratin 9 [Homo sapiens] gi|119617047 keratin 2A (epidermal ichthyosis bullosa of Siemens) [Homo sapiens] gi|119581084 keratin 10 (epidermolytic hyperkeratosis; keratosis palmaris et plantaris), isoform CRA_a [Homo sapiens] gi|122920512 Chain A, Human Serum Albumin Complexed With Myristate And Aspirin gi|119587944 heat shock 70kDa protein 8, isoform CRA_a [Homo sapiens] gi|386785 heat shock protein [Homo sapiens] gi|194388088 unnamed protein product [Homo sapiens] gi|292059 MTHSP75 [Homo sapiens] gi|158255142 unnamed protein product [Homo sapiens] gi|47940601 Keratin 5 [Homo sapiens] gi|63100331 Keratin 14 [Homo sapiens] gi|410171504 PREDICTED: hornerin [Homo sapiens] gi|1143492 BiP [Homo sapiens] gi|31074631 keratin 1b [Homo sapiens] gi|119617032 keratin 6B, isoform CRA_a [Homo sapiens] gi|119600472 protein disulfide isomerase family A, member 4, isoform CRA_a [Homo sapiens] gi|38013966 TKT protein [Homo sapiens] gi|687237 tumor necrosis factor type 1 receptor associated protein, partial [Homo sapiens] gi|148271059 dermcidin [Homo sapiens] gi|119581157 keratin 17, isoform CRA_d [Homo sapiens] gi|3983129 desmoglein 1 [Homo sapiens] gi|31873640 hypothetical protein [Homo sapiens] gi|74096732 filaggrin 2 [Homo sapiens] gi|119617046 keratin 6 irs3 [Homo sapiens] gi|184761 immunoglobulin alpha-2 heavy chain [Homo sapiens] gi|21755908 unnamed protein product [Homo sapiens]

Supplementary Table S8 - Primers and probes used for quantitative PCR

Name Accession No. Forward primer (5’-3’): Reversed primer (5’-3’): UPL#

β2-microglobulin NM_004048.2 TAGGAGGGCTGGCAACTTAG CCAAGATGTTGATGTTGGATAAGA 42 ribosomal protein NM_000992.2 CAGCTCAGGCTCCCAAAC GCACCAGTCCTTCTGTCCTC 53 L29 CEBPA NM_004364.3 GGAGCTGAGATCCCGACA TTCTAAGGACAGGCGTGGAG 28 CEBPB NM_005194.2 GCGACGAGTACAAGATCC AACAAGTTCCGCAGGGTG 74 MPO NM_000250.1 TTCGTCACTGGCGTCAACT ATTGGGCGGGATCTTGAG 38 ELANE NM_001972.2 TTCCTCGCCTGTGTCCTG CTGCAGGGACACCATGAA 3 GCSFR NM_000760.3 AGCGCGAGCAATAGCAAC TGAAAGGGCCTGATGTTCTC 23 AGTCTCCGAGAGAAGAAAAG NM_001161529.1 CGGATTTTCCTGCTGTAAACC GMCSFR CA 12 HK3 NM_002115.2 CCTCCCACTGGGTTTTACCT GAAACCCTTGGTCCAGTTCA 37 SOX4 NM_003107.2 AGCCGGAGGAGGAGATGT TTCTCGGGTCATTTCCTAGC 20

Supplementary Table S9 - Antibodies used for Western blot analyses

Antibody target Source and cat. no. Manufacturer Working concentration PDI (C81H6) rabbit Cell Signaling 1:1000 mAb #3501 actin-HRP conjugated mAb A3854 Sigma-Aldrich 1:50,000 α-Tubulin (DM1A) mouse mAb Calbiochem 1:1000 CP06 C/EBPα rabbit polyclonal ab15048 Abcam 1:1000 HDAC2 rabbit polyclonal#2540 Cell Signaling 1:1000 Thioredoxin 1 (C63C6) rabbit mAb Cell Signaling 1:1000 #2429 Biotin-HRP goat #7075 Cell Signaling 1:1000 conjugated GAPDH (14C10) rabbit Cell Signaling 1:1000 mAb #2118 ERO1α rabbit polyclonal #3264 Cell Signaling 1:1000 XBP1 (D2C1F) rabbit Cell Signaling 1:1000 mAb #12782 Phospho-eIF2α (L57A5) mouse Cell Signaling 1:1000 mAb #2103 BiP (C50B12) rabbit Cell Signaling 1:1000 mAb #3177

Supplementary Table S10 – shRNA used for PDI and TXN knock-down

Catalog no. Target shRNA sequence Non- SHC002V Mammalian Not known shRNA Control CCGGCGAGTTCTTTGGCCTGAAGAACTCG TRCN0000049193 PDI (a) AGTTCTTCAGGCCAAAGAACTCGTTTTTG CCGGGTGTGGTCACTGCAAACAGTTCTCG TRCN0000049194 PDI (b) AGAACTGTTTGCAGTGACCACACTTTTTG CCGGGTGTGGTCACTGCAAACAGTTCTCG TRCN0000290651 PDI (c) AGAACTGTTTGCAGTGACCACACTTTTTG CCGGCGACAGGACGGTCATTGATTACTCG TRCN0000049196 PDI (d) AGTAATCAATGACCGTCCTGTCGTTTTTG CCGGTGCTGTTCTTGCCCAAGAGTGCTCG TRCN0000296736 PDI (e) AGCACTCTTGGGCAAGAACAGCATTTTTG CCGGGCTTCAGAGTGTGAAGTCAAACTCG TRCN0000064279 TXN (a) AGTTTGACTTCACACTCTGAAGCTTTTTG CCGGGCAGGTGATAAACTTGTAGTACTCG TRCN0000064278 TXN (b) AGTACTACAAGTTTATCACCTGCTTTTTG CCGGGATGTGGATGACTGTCAGGATCTCG TRCN0000064280 TXN (c) AGATCCTGACAGTCATCCACATCTTTTTG CCGGATCAAGCCTTTCTTTCATTCCCTCGA TRCN0000369941 TXN (d) GGGAATGAAAGAAAGGCTTGATTTTTTG CCGGGCTGGAGCTGACCAGTGACAACTCG TRCN0000007305 CEBPA (a) AGTTGTCACTGGTCAGCTCCAGCTTTTT CCGGCAAGAAGTCGGTGGACAAGAACTCG TRCN0000007307 CEBPA (b) AGTTCTTGTCCACCGACTTCTTGTTTTT CCGGCCCGGCAACTCTAGTATTTAGCTCG TRCN0000356198 CEBPA (c) AGCTAAATACTAGAGTTGCCGGGTTTTTG

Supplementary Table S11 – AML patients data

Blasts in WBC RBC PLT Neut Age at Molecular Hgb [g/ No Diagnosis Sex FAB Karyotype bone [×103/ [×106/ [×103/ [×103/ diagnosis signature dl] marrow µl] µl] µl] µl]

chr. 7 Pt 1 AML/ MDS 36 F 40 19.45 2.8 8.7 50 45.5 monosomy AML- NPM1 normal Pt 2 AML 43 F 39.54 4.15 12.6 143 -- M5 mutation karyotype NPM1 chr. 8 Pt 3 AML 59 M 41.07 4.69 11.5 26 -- mutation trisomy AML- Pt 4 AML 27 M 66.02 4.28 10.4 34 9.11 M4 mixed Pt 5 25 F 14.7 2.92 5 22 -- karyotype AML atypical AML AML- Pt 6 (CML->AML- 58 M 28 M4 M4)

Abbreviations used: AML – acute myeloid leukemia; chr – chromosome; CML – chronic myeloid leukemia; F – female; FAB – French-American-British AML classification; Hgb – hemoglobin; M – male; MDS – myelodysplastic syndrome; Neut – neutrophils; PLT – platelets; RBC – red blood cells; WBC – white blood cells.

Descriptions to supplementary figures and a video file

Supplementary Fig. S1. SK053 exerts anti-leukemic effects and induces differentiation of AML cell lines in vitro. A, MOLM14, NB4, and KG1 cells were incubated with serial dilutions of SK053 for up to 5 days. Each day the number of dead cells was evaluated with trypan blue exclusion. Graphs present mean percentage of dead cells comparing to controls ±SD; *P<0.05 vs controls, one-way ANOVA with Dunnett’s post-hoc test, n=6. B, MOLM14, NB4, and KG1 cells were incubated with serial dilutions of SK053 for up to 5 days. Each day the number of living cells was evaluated with trypan blue exclusion. Graphs present mean cell number ±SD; *P<0.05 vs controls, one-way ANOVA with Dunnett’s post-hoc test, n=6. C, Semi-quantitative colorimetric NBT reduction assay in MOLM14, NB4, and KG1 cells incubated for 5 days with indicated SK053 concentrations; graphs present mean % increase vs control ±SD; *P<0.05 vs controls, one-way ANOVA with Dunnett’s post-hoc test, n=3.

Supplementary Fig. S2. Diagram of RNA sequencing data analysis pipeline used for differential gene expression (DGE) analysis in the SK053-treated HL-60 cells.

Supplementary Fig. S3. Differential gene expression (DGE) analysis of HL-60 cells treated for 48 h with 10 µM SK053 vs DMSO treated controls. A, Volcano plot of genes differentially expressed between the cells treated for 48 h with SK053 and control cells (DMSO). B, Diagram of significantly enriched Biological Process GO nodes corresponding to the results of DGE results obtained for cells treated for 48 h with 10 µM SK053. The yellow and orange nodes represent terms with significant enrichment, with different color shades representing various degrees of significance i.e. from 1.00E-2 (yellow) up to <1.00E-6 (red). C, Diagram of significantly enriched Molecular Function GO nodes corresponding to the results of DGE results obtained for cells treated for 48 h with 10 µM SK053. The yellow and orange nodes represent terms with significant enrichment, with different color shades representing various degrees of significance i.e. from 1.00E-2 (yellow) up to <1.00E-3 (red).

Supplementary Fig. S4. Differential gene expression (DGE) analysis of HL-60 cells treated for 120 h with 10 µM SK053 vs DMSO treated controls. A, Volcano plot of genes differentially expressed between the cells treated for 120 h with 10 µM SK053 and control cells (DMSO). B, Diagram of significantly enriched Biological Process GO nodes corresponding to the results of DGE results obtained for cells treated for 120 h with 10 µM SK053. The yellow and orange nodes represent terms with significant enrichment, with different color shades representing various degrees of significance i.e. from 1.00E-2 (light yellow) up to <1.00E-7 (orange). C, Diagram of significantly enriched Molecular Function GO nodes corresponding to the results of DGE results obtained for cells treated for 120 h with 10 µM SK053. The yellow and orange nodes represent terms with significant enrichment, with different color shades representing various degrees of significance i.e. from 1.00E-2 (light yellow) up to <1.00E-7 (orange).

Supplementary Fig. S5. SK053 induces ER stress in HL-60 cells. Representative Western blot results showing UPR and ER stress markers induction in HL-60 cells upon incubation with 10 µM SK053, GAPDH levels served as a loading control.

Supplementary Fig. S6. Evaluation of cell growth and differentiation in HL-60 and MOLM14 cells upon TXN knock-down. A, Control HL-60 cells and HL-60 cells transduced with non- targeting (NTC) or TXN [TXN(a)]-targeting shRNA lentiviral particles were counted daily in trypan blue from day [d] 1 till day 10. B, Western blot showing the efficacy of TXN knock- down in HL-60 and MOLM14 cell lines transduced with TXN-targeting shRNA [(TXN(b) or TXN(c)]-encoding lentiviruses. GAPDH served as a loading control. C, Growth of HL-60 cells transduced with non-targeting (NTC) or TXN-targeting [(TXN(b) or TXN(c)] shRNA. Cells were counted daily in trypan blue from day 1 till day 9. *p<0.05 vs NTC in two-tailed Student’s t test. D, Growth of MOLM14 cells transduced with non-targeting (NTC) or TXN- targeting [(TXN(b) or TXN(c)] shRNA. Cells were counted daily in trypan blue from day 1 till day 9. *p<0.05 vs NTC in two-tailed Student’s t test. E, Mean percentage ±SD of AML cells modified with non-targeting (NTC) or TXN-targeting [(TXN(b) or TXN(c)] shRNA, expressing CD11b myeloid marker determined in flow cytometry, N=4, p values (p) calculated vs NTC, two-tailed Student’s t test, NS-not significant.

Supplementary Fig. S7. Evaluation of cell growth and differentiation in HL-60 and cells upon CEBPA knock-down A, Western blot showing C/EBPα levels in two distinct sets (1 and 2) of HL-60 cells transduced with non-targeting (NTC) or PDI-targeting [PDI(d)] shRNA. GAPDH served as a loading control. B, Western blot showing the efficacy of CEBPA knock-down in HL-60 cells transduced with CEBPA-targeting shRNA [CEBPA(a) or CEBPA(c)]-encoding lentiviruses. GAPDH served as a loading control. C, Growth of HL-60 cells modified with CEBPA [CEBPA(a) or CEBPA(c)]-targeting shRNA and incubated with 5 or 10 µM SK053. Cells were counted daily in trypan blue from day 1 till day 8. D, Mean percentage ±SD of HL- 60 cells modified with non-targeting (NTC) or CEBPA [CEBPA(a) or CEBPA(c)]-targeting shRNA, expressing CD11b myeloid marker determined in flow cytometry, N=2.

Supplementary Fig. S8. Chemical structures of compounds. A, Chemical structure of SK053. B, Chemical structure of SK-BIO – a biotinylated SK053 analog. C, Chemical structure of SK-IN – an inactive, biotinylated SK053 analogue that lacks the electrophilic double bond. D, Chemical structure of SK053 showing the localization of the leaving group and truncated form of the compound. E, Potential mechanism of SK053 binding to catalytic cysteines in PDI, 1 or 2 truncated SK053 molecules bind to PDI catalytic site.

Supplementary Fig. S9. Proteomic assessment of SK053 binding site in the human PDI (huPDI). A, Amino acid sequence of huPDI showing peptides identified by mass spectrometry (bold red). The peptide containing Cys residue with SK053 modification is underlined. (b-d) Example MS/MS data for the peptide 400KNVFVEFYAPWCGHCK415 from huPDI. MS/MS fragmentation was performed on a Thermo Orbitrap Velos coupled with Waters nanoAcquity UPLC and data were analyzed by Mascot software. This peptide had a

Mascot ion score of 56, precursor mass of 2209.054, charge state of +3. B, The MS/MS fragmentation spectrum with y-series and b-series ions labeled. C, Table of detected masses as annotated by Mascot. Bold italic red indicates that the ion series contributed to the peptide score. Bold red indicates the number of matches in the ion series is greater than would be expected by chance alone, suggesting that the ion series is present in the spectrum. Non- bold red means that the number of matches in the ion series is no greater than would be expected by chance. Masses indicated in black type were not detected in the spectrum. C* indicates the most probable site of SK053 modification on Cys414. D, Alternate Mascot site analysis indicates another putative SK053 binding site on residue Cys411 (marked as C#). E, SK053 binds to the 4th thioredoxin-like domain of huPDI (PDIA1-d4): the recombinant PDIA1- d4 protein was incubated without (left) and with SK053 in a molar ratio 1:10 (right). The mass shift observed in the right panel corresponds to two molecules of the inhibitor after the elimination of the leaving group (2 × 282 Da), according to the mechanism described previously.3

Supplementary Fig. S10. Covalent docking of SK053 to two cavities in the 4th thioredoxin- like domain in human PDI. A, Truncated SK053 molecule (molecule without the leaving group, Supplementary Fig. S6d) bound to Cys397 of huPDI. The polar contacts between the ligand and the protein are shown with a dotted, yellow line. The ligand is placed in the “primary” cavity. B, Truncated SK053 molecule bound to Cys400 of huPDI. The polar contacts between the ligand and the protein are shown with a dotted, yellow line. The ligand is placed in the “secondary” cavity. C, Two truncated SK053 molecules bound to either Cys400 or Cys397of huPDI. The ligand can be either placed in the “primary” cavity or the “secondary” cavity.

Supplementary Video S1. Two putative binding sites of hPDI are located on the opposite site of the helix harboring active site cysteines. Molecular docking with GOLD suggests that both ‘primary’ binding site (depict cyan on the protein surface) and ‘secondary’ binding site (magenta) are equally probable to host SK053 molecule. Full atom representations of both binding sites are presented in Supplementary Fig. S10.

120 MOLM14 120 NB4 120 KG1 SK053 SK053 100 100 * [µM] 100 SK053 * * [µM] [µM] 80 80 * 0 80 * 0 2.5 0 60 5 * 60 60 5.0 10 10 * * 40 * 7.5 15 * 20 40 * * 40 * * * % of dead cells * * * 20 * % of dead cells * % of dead cells * * 20 * * * 20 * * 20 * * ** 0 0 0 24 48 72 96 120 24 48 72 96 120 24 48 72 96 120 time of incubation [h] time of incubation [h] time of incubation [h] B 3 0.8 SK053 SK053 SK053 [µM] 2.0 [µM] 0.6 [µM] 2 0 0

] 0

] 1.5 6 6 ] 6 2.5 5 0.4 * * 10

* 10 10 * 10

5.0 1.0 ´ ´ [ [ ´ 10 15 [ 1 * 7.5 * * 0.2 0.5 20 20 0.0

0.0 Number of viable cells 0 Number of viable cells Number of viable cells 0 24 48 72 96 120 24 48 72 96 120 0 24 48 72 96 120 time of incubation [h] time of incubation [h] time of incubation [h]

C 100 500 500 80 * 400 400 * 60 300 300 40 200 200 * 20 * 100 NBT reduction NBT reduction 100 NBT reduction [% over controls] [% over controls] * [% over controls] * 0 0 0 ctrl SK053 SK053 ctrl SK053 SK053 ctrl SK053 SK053 5.0 μM 7.5μM 2.5 μM 5.0 μM 10 μM 15 μM

Supplementary Figure S1 Supplementary Figure S2 70 HMGCS1l A S100A9l 60

l UNC5B 50 IDI1l

l CLGN TUBA1Al S100A8l

DBIl 40

CLUl

UBL5l HMGN3l

(FDR) l TRIB3

IL1B

10 l UCP2l

C6orf223 ELOVL6l l

GLIPR1 PLXNA1 INSIG1l l

30 l

RBCK1 EBP CD86 ALDH1L2 l ANXA1l l l l SYTL1 SPNS3 l l HMGCRTMSB10l CETN2l l −log l MT1X SELL CLC MNDAl l l ACAT2l

l CDT1 CD53l CYTIPl CD59l MFAP4 l PAK1l l l PKD1 MGAMl CHRM3−AS2 l l l CXCR2C1orf186l DlYNLL1l l CCL2l C14orf1 RPS26 l 20 TBC1D16 l l l MGST1l GPR34 l HSP90AA1 l NFKBIL1 SQLE l LSS l l l lADI1 l l l CENPIl P2RY14 ITGAM l SLIRPl ADGRE3 . SNHG7 l l l l l ASNS ll HSPE1 l l l l l l NDUFA3l l CCNB1IP1 l l l BTG2 SNHG1 l l l ll l llCYP1B1 GNG2 l FPR1lHMGN5KCTD12 l l l l PECAM1l l l NDUFC1 lCD84l PHLDA1 LILRA5 l l l l l AGPAT9 l l l l l l PHGDH l PIR l l SRD5A3 l l l DNASE2l l l CLEC5A EGR1AMICA1 EPB41L4A−AS1l l l F5 CTSG l l l l lCARD9 DYNLl T1 MPZL3 l l l l l l l POLR2LSGK1CR1 FABP5 l l l l PCK2 ll l l LRRK2 l l l l l l l l l l l l ll l l l l l l l l l l l l SPSB3 l l KLHDC4l l ll l PIK3R3 SIGLEC7 TCEA3l l ll lSESN1 l l ll l ARRDC3l l l l l STS l l l l GBP2 AZU1 l l l l DOCK10l TMEM2DTX4l LILRB2 l l l l l l GAB2l l l l VLDLR−AS1 l l ll BCL6 l MIB2 l ll SESN3l l l l ll l CRYZl l l l l l l l l ll SAMHD1 l l l l l l RCBTB2l l SH3BP5l l FFAR2 l l ll PFN2CD52l l l l l AQP9 l l l l ll CD300Cl l l l l ll l l l lSLC16A13l l l ll ll l ll l l l l l MED31LILRA1DDX60LDLEU2 l 10 KLHDC7B ELFN2 ll l l l l l l HSP90AA2P GDAP1L1 l l l l l l l l l l ll SERPINB8l l l l MPEG1. l l l ll l lll l l l l LYZ TLR1 l IL1RL1 l l l l ll ll l l ll l l l l l l l l l PCED1B−AS1l FAIM3 l l l FKBP9 l l l l l TNF l l CCR3 l l l l l l l l RGS16 l l l ll l l l l SPP1 l l lll l l l ll l l lll l l l l l l l l l TMEM71l l GNB1Ll HLA−A l l l ll l l l l l ll l l l lCYP4F3l l l l l l llll lll l l l B4GALNT1FOSEGR2 l l llll l l l l l l ME1l SLC40A1 LRRC75A INHBE l l l l l l l l l l l l l IFIT2 l l l ll l l l lll l l l ll l l l l l l l l llll l l l l l l l l l l l SCML1 l l l l l l l l l ll l . CEACAM1 l l l l l l l l l l l l l l ll ll l l ll l l l FBP1 l l . l l l l l l l l NAT1 l l l l l llll ll l KIAA0513l l KDM7A l l l l l l l l l l l . SGMS2 . C2orf48 l l l l l l l ll l l l l l l INPP4B BATF2 l l l l l l l l ll l l ll ll l IL18BP l l l ll l ll l l llSAMD9LKIAA0226L l l l HIST3H2A l l l l l l ll l l l TM7SF2SIGLEC15 DENND2D NDUFA4L2 l ll ll l l l l l l ll ll l l l TRIM22 l l l ll l l lll ll l l l l l l l l ll ll l lll l llll l l l l l l l l lll l l l CAMSAP2 l l ll l l l ll l ll l ll l ll CCDC167S100A12 l l l l l ll l l l ll ll l OSBPL6 l l ll l llll ll l l lll l ll l l l l l l llll l l CREB5l l l l l l l l l l l l lll l l ll l l l . LILRB1 DAAM2 l l l l l l l l l MAP2K6 ll l l l l ll l l l ll l l l l l l RHOB ADRB2 TNC LHX4 ll l l l l lll ll l lllll ll llllll l l l CSF1RKLF2 l l l l l l l ll l l l IFIT3l l NBPF17Pl l l l l ll l l l ll l l l l l ll l llll l l ll HPGDFAM65Bll l SIGLEC9l . l ll l l l l ll l l l l l l l ll ll l lll l l ll l l l l MYO5C l l l l l l l l ll l l l ll l l l l CSGALNACT1l l VASH2 l ll l l l l l ll l ll l l ll SNRPGP10l l . l l l CCL23. l KIF26B NAV1STOX2 ll l l lll l ll l l llllll ll l l lll l l l l ATP10D l l l l ll l l l ll l lll l l PFN1P1 CCDC64 l l ZNF296 l l l ll l l l l l ZFP36L1l SLC8A1 l l l l lGPR85 l l l l l l ll l l l l l LILRB3 l l ll l l l l l lll l l l l l l l l ll HERC6l CCL22 CCL3L3l. DLK1 l l ll l l l l l ll lll l l l l l TAF9B l l . PARK2 l l ll l l ll l ll l l l l ISM1 l l l l l PTGES l l l l l l l l ll l l l l l l l PKD1P6 l l ll l l l l l l l l l l l l l l l l l l l l l l l l l LINC00174. l l l l l l l l l l l l l lll l l l l SLC16A6l MILR1CLEC4A l . ll l ll l l ll lll l llll l l l l FAM3C . l N4BP2L1l l l l l l lll l l l l lll ll l l ll llll l l l IPP l . TMEM86B l l l l l l l CYP4V2APOLD1l l ll ll l ll l l ll l l l l l . SPTBl l l l l l l l l ll lll l lllll ll l l l THAP7−AS1 ALDH2 STMN3 l l ll lll ll l l l ll l ll l llll l l l lllll l l l ll l l l ll ll EPS8 CXCL16. ITGA2B l l l l l l l l TLR8 VSIG4 l l l l l l l l l l l l l l l l lll l l l HSPE1P2 l . l l l llll lll lll lll lll ll l l l ll KIAA1598 HSPE1P3 l l l l SIGLEC17P . ZNF767P l l ll lll ll l SEPSECS−AS1l l l MST1 ll l l l l l l l l ll l l l lll ll ll l ll l l . l l l l l l l l IGSF6 l JPH2 l l l l l l l l l l l l l l l PTPRO l l l l ll l l l l l l l l l l WDR27l l l l l l l POLR3G l l l l l l l l l l l l l l l l l l l ll l l l l l l ll l l ll l l TTC28C1QTNF6 ll l l l l ll ll l l l l l l l l NOP56P3 CYP26A1 l l l l l l l l l IFIT1 l l l l l . l l l l l l l l l l l l l l l l PPM1M l l l HIST1H3H l l l l l l l l l l l ll ll ll l l l l l l ll llll l l l l l CBS l l . SNORD116−4 PCSK4 l l l l l l l l l l lll l l l l UBL5P2 . CCDC30C21orf2l l l ll ll l l ll l ll ll llllll l l l ll l l GCNT2 PRKAR2B TRPM4 l SLFN12ANKRD24l l l l l l l lllll l l l l l l l ll l lll lllll l l l l lll l l l l l l l l l HMGN2P8 l l l l l l l lll ll ll l l l GPR82SRGAP3. l l l l l l l l l l l S100Z l l SH3D19 l l l l l l l l l l l l l l l l ll l l l l . .l NOL12PLXDC1RAPGEF3 ll l l lll l l l l ll l l l ll l ll l l l lll ll l llll l l ll l ll l l lll ll l l PFKFB2l l l l l l ll l ll l l l l ll l l l l l l l ll lll l ll l l l l . l MKLN1−AS MTG1 l l l l l l l ll l l l l l lARHGEF3 SDCBP2 l ll ll llll ll l l ll l l l l l PXN l . C5orf66 l l l l l l l l C9orf3 l l . l. l ll l ll l l l l ll ll lll lllll l l l l l LILRA6 l l l ll l l l l l l l FCRL1 l l l l l DSC2 HIC1 . . l ll l l l ll l l ll ll l l ll l l l l l l l l l ll l l l l l l l l ll l l l l l l l l . GPR176 GOLGA6L9 l l l l l l l l l l l l l l l ll l l l l . l l l l l OAS3 l l l l l l l l l . ACAD11 l l l l l l l l l l CTTNBP2NL SRGAP1 l l l l l l l l l l l l l l l l l l MBNL1−AS1 l ll l l l l l l l l l l l SCN4B PBX1 RHBDL1 SNX22 l l l l l ll l l ll l DOCK3l l l l l l ll l l l l l l RFPL3S l l l l l l l l l l l l FKBP9P1. l ll l l l l l l ll llll l l l l l l l l l ll l l l l l l ll l l l l l l l PRSS57GAD1.l l l l l l l l l l l lll l ll ll l LIME1 l l l l l l l l l l l l l l ll l l l l l l l l l . l . l ll l ll l l l l l l l l l ll ll l l ll l l l . l l ll llll l l l l ll l l l ll ll l l l l l l l l MREG. CALHM1 TRIM67 l l l l CD1D ATP6V0E2−AS1l l l l ll l l lll l l l GNGT2. . l l l ll l l l l l l l l l l l l l ll ATP5EP2 l l .HCG4P3 l l l l l l l l l ll ll l ll llll l l l ll CEACAM19l HSPG2 .ANKDD1Al l l l ll ll l l l lll l l llll ll ll l SLAMF6l JAZF1−AS1IL1A . l l l l l l l l RN7SKMAFB. CADM1 lLY6G5BCAPS l l l l l l l l . SIX5 TPPP l l ll l l ll l l EPX l l l l l l l l l l l l l l l l l l l l l l l l l l NGFR . l l l l l l l l l ll l l ll l ll l l ll l l l l ll l l l l l l ll l ll l l l l l l l l l l l ll ll l l l l ll l l l l ll l l lll l l ll ll ll l ll l ll lll l l lll ll l l l l ll l l l l ll l l l l l l l l l l l l l l l ll l ll l l l l l l l l lll l l l l l l l l ll l l l l l l l l l l ll l l l llll l ll l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l l ll ll l l l l l l l l l lll l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l ll l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l ll l l l l l l l l ll l l l l l l l l l l l l l l l l l l ll l l l l llll l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l ll l l l l l ll lll l ll l l l l ll l l l ll l l l l l l l l l l l l l l l ll l ll l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l ll ll ll ll l l l l l l l l l l l l l l l l l l l l l ll l ll ll l l lll l l l l lll lllllllll lllllllll l ll l l l l l l l l l l l l l l l l l l l l l l l l l l llll ll l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l ll l ll l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l lll l l l ll l l ll l l l l l l l l l l l l l l l l l l l l l ll lll l l l l l l l l lll l l l l l l l l l l l l l l l l l l l l l ll l lll l lll l l l l l l l l l l lll ll l l l l l l l l l l l l l l ll ll l l ll l lll ll l l l l l l l l l l l l l l l l l l l ll l ll l ll l l ll l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l ll ll ll l ll ll l ll lll ll l l l ll l l l l l l l l l ll l ll l l l l l l l l ll l l l l l l l l l l l l l l l l l ll l l l l l l lllll l l l l l l l l l l ll l l l l l l l l l l ll l l l ll l l l l l llllll l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l 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l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l ll l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l ll l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l lll l l l l l l l l lll l l l l ll l l l l l l l l ll l l l l l l l l l l l l l l l l ll l l ll ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll ll l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l lll l l l ll l ll l l l l l l l l l l l l ll l l l l l l l ll l ll l l l l l lll l l ll l l l l ll l l l l l l l l l l ll l ll l l l l l ll l l l ll l l l l l l ll l l l l l l l l l ll l l ll l l l l l l l l l l l l l l l l l 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l l l l l ll l l l l l ll l l l l l l ll l l l l l l l l l l l ll l ll l l l l ll l l lll l l ll l l l ll l l l l l l l l l l l l l l l ll l l l l l ll ll l l l l l l l l l l l l l l l l l lll l l l l l l l l l l l l ll l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l ll l l l ll ll ll l l l l l l l l l l l ll l ll l ll ll l l l l l ll l l l l l l l l l l l l l l l ll l ll l l l l l l l l l l l l l lll l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l l ll lll l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll ll l ll l l l ll l ll l l l l l lll ll l l l l l l l l l l l l l l l l l ll l l l l l l l ll llll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l lll l l l ll l l l ll l l l l l l l l l l l l l l l l l l l l l l llll l l l l l l l ll l l l l l l l l l ll ll l l ll l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l l ll ll l l l l l lll l l l l l ll l l ll l l l l l ll l l l l l l l l l l l l ll l l l l l l l l l l ll l l l l l l l l lllll l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l ll l l l ll l l l l l l l l l l l l lll l ll l ll ll l l l l ll l l l ll ll l l l l l l l l l l l l l l l l ll l l l l ll l ll l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l ll l l l l l l ll l l l l l l l l l l l l l l l l ll l l l ll l l l l l l l l ll lll l l ll l l l l l l l l ll l l ll l l l l l l l l ll l l l l l lll l ll l l l l l l l l l l l l ll l l ll l l l ll l lll ll l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l ll l l lll l ll l l l ll l l l l ll l l l l l l l l l l l l l lll l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l ll l l lll l l l l l l l l l l l l l l l l l l lll l ll l l l l l l l l l l l ll l l l l l l ll l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l ll l l l l ll l l l l l l l l l l l ll l l ll ll l ll l l l l l l l l ll l l l l lll l ll l l l l l l l l ll lll ll l l l l l ll ll l l l l l ll ll l l l l l l l l l ll l l ll l l l l l l l l l l l l l l l l ll ll l l l ll l llll llll l l l l llll l l l l l l l l l ll l l ll l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l ll ll l l l l l l l l l l l l l l l ll ll l l l l l l l l l l ll ll l l l l l l l l l l l l l l ll l ll l l ll l l l l l ll l l lll l l llll ll l l l l ll l l l l l l l l ll l l l l l l l l l l ll l l l l l l l ll l l l l l l l l l l l l l l ll ll l l l l l l l l l lll l l l l l l ll l l l l l ll ll l lll l l lll l l l l l l l ll l l l l l l l l l l ll l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l ll l l l l l l l l l l l l l l l l ll l l l l l l l l ll ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l ll l l l l l l l l ll ll l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l llll ll ll l l l l l ll lllll l l l ll l l l l ll l l ll ll l l l ll l l l l l l ll lll l l l l l l l l l l l l l l l ll l l l l l l l l l ll l l l l l l l l l l l l l l l ll l l l l l l l l l ll ll l l l l lll l l l l l ll l l l ll l l l l ll l ll ll l ll l l l l l l l l lllll l l l l l l ll l ll l l l ll l l l ll l l l l l l ll l l ll l l l ll l l l l l l l ll l l l ll ll l l l l ll lll l l l lll l l l l l l l l l ll l l l l l l ll l l l l l l l l l l l l l ll l l ll l l l ll l l ll l l l l l l l l l l l ll l l l l l l l ll l l l l l ll l l l l l ll l l ll l l ll l ll l l l l l l l l l l l l l ll l l l l l l l l l l ll l l ll l l ll l ll l l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l ll l l ll l l lll l l l l l l l l l ll ll ll l ll l l ll l l l l l ll l l l l l l l l l l l l l l l l l l ll ll l l ll l l ll l l l l l l ll l l l l l l l l ll ll l l l l l l l l l l l l l lll ll ll l l l l l ll ll ll l l l l ll l l l l l ll l l l l l l l l l l ll l l l l l ll l ll l l ll l l l l l l ll ll l ll l l l l l ll l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l ll l l l l l l l l ll l lll l l l l l l l ll l l l l l l l l l l l l l ll l l llllll l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l ll ll l llll l lll ll ll ll l l l l l l l l l l l l l l l l ll l l l l l l l l ll l ll l l l l l l l l lll ll l l l ll l l l l lll l ll l l l l l ll l l ll l l l l ll l l l l l l ll l l l l l l l l l l l l l l l l l l l l l ll ll ll l l l l l ll l l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l lllll l l l l l l l l l l l l l l l l l lll l l l l l l l l ll l l l l l l l l l l ll l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l ll l l l l ll l l l l l l l l l l l ll l l l l l ll l l l ll l l ll l l ll l l l l l l ll l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l ll ll l l ll l l l lll l l l l l l l l l l l l llll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l lll l ll l lll l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll ll l l l l l l l l l l lll l l l l l l l l l l l l l l l l l l l l l l l ll l l ll l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l ll l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l ll l ll l l l ll l l l l l l l l l l l l l l l l l l l ll l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l lll l l l l l l l l l l l l l l l l l l l llll l l l l ll l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l lll ll l l ll l l l l l l l l ll l l l l l l l l l l l l l l l ll l ll l l l l l l l l l l l l l l l l l l l l l ll ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l lllll l l l l l l l l l l l l l l l l l l l ll l lll 0

−6 −4 −2 0 2 4 6

log2(sk.48h_vs_ctl.48h)

GO:0008150 biological_process 1.000000 1287 / 5486

GO:0008152 GO:0065007 GO:0009987 GO:0044699 GO:0032501 GO:0023052 GO:0051179 GO:0051704 metabolic process biological regulatio... cellular process single−organism proc... multicellular organi... signaling localization multi−organism proce... 1.000000 1.000000 1.000000 5.25e−06 0.110857 0.000656 1.000000 1.000000 B 922 / 4062 836 / 3447 1183 / 5011 1085 / 4299 500 / 1865 501 / 1804 441 / 1732 194 / 734

GO:0044238 GO:0071704 GO:0009058 GO:0044710 GO:0044237 GO:0002376 GO:0050789 GO:0044763 GO:0016265 GO:0044707 GO:0044700 GO:0050896 GO:0051674 primary metabolic pr... organic substance me... biosynthetic process single−organism meta... cellular metabolic p... immune system proces... regulation of biolog... single−organism cell... death single−multicellular... single organism sign... response to stimulus localization of cell 1.000000 1.000000 1.000000 1.000000 1.000000 2.17e−07 1.000000 0.002228 0.104352 0.035948 0.000656 1.84e−07 1.000000 819 / 3728 839 / 3822 489 / 2249 507 / 1947 852 / 3776 269 / 823 794 / 3287 990 / 3922 229 / 776 491 / 1812 501 / 1804 695 / 2523 97 / 353

GO:1901360 GO:0006629 GO:1901576 GO:1901615 GO:0044711 GO:0044281 GO:0055114 GO:0006091 GO:0048518 GO:0002682 GO:0001775 GO:0048583 GO:0006955 GO:0050794 GO:0008219 GO:0007154 GO:0051716 GO:0006950 GO:0042221 GO:0009607 GO:0009605 GO:0040011 GO:0006928 organic cyclic compo... lipid metabolic proc... organic substance bi... organic hydroxy comp... single−organism bios... small molecule metab... oxidation−reduction ... generation of precur... positive regulation ... regulation of immune... cell activation regulation of respon... immune response regulation of cellul... cell death cell communication cellular response to... response to stress response to chemical response to biotic s... response to external... locomotion cellular component m... 1.000000 1.000000 1.000000 1.000000 1.000000 0.084838 0.011128 0.038557 1.000000 0.003407 3.61e−05 0.585450 7.88e−07 1.000000 0.091350 0.001442 0.001770 0.001246 0.000545 0.078336 1.44e−05 1.000000 1.000000 523 / 2490 130 / 437 477 / 2218 51 / 165 155 / 537 323 / 1143 88 / 240 70 / 185 392 / 1485 141 / 419 120 / 321 300 / 1075 174 / 488 757 / 3137 229 / 775 504 / 1825 563 / 2071 364 / 1259 332 / 1125 86 / 243 206 / 619 137 / 475 153 / 537

GO:0008202 GO:1901362 GO:0008610 GO:1901617 GO:0006066 GO:0044283 GO:0022900 GO:0015980 GO:0002684 GO:0045321 GO:0050776 GO:0010941 GO:0012501 GO:0007165 GO:0006952 GO:0009611 GO:0070887 GO:0010033 GO:1901700 GO:0043207 GO:0042330 GO:0048870 steroid metabolic pr... organic cyclic compo... lipid biosynthetic p... organic hydroxy comp... alcohol metabolic pr... small molecule biosy... electron transport c... energy derivation by... positive regulation ... leukocyte activation regulation of immune... regulation of cell d... programmed cell deat... signal transduction defense response response to wounding cellular response to... response to organic ... response to oxygen−c... response to external... taxis cell motility 0.136857 1.000000 1.000000 1.000000 1.000000 0.013742 0.000787 0.004585 0.006156 0.007202 0.011781 0.026802 0.050281 0.000721 6.57e−07 0.002817 0.527229 0.078336 0.020934 0.046377 0.020934 1.000000 40 / 93 324 / 1597 73 / 224 30 / 72 47 / 139 68 / 174 36 / 68 60 / 144 93 / 254 86 / 231 101 / 285 165 / 521 210 / 696 469 / 1674 178 / 501 138 / 407 222 / 766 247 / 843 138 / 420 83 / 230 78 / 209 97 / 353

GO:0016125 GO:0006694 GO:0046165 GO:0045333 GO:0043067 GO:0006915 GO:0045087 GO:0014070 GO:0006935 GO:0016477 GO:0051707 sterol metabolic pro... steroid biosynthetic... alcohol biosynthetic... cellular respiration regulation of progra... apoptotic process innate immune respon... response to organic ... chemotaxis cell migration response to other or... 0.397110 0.016358 0.130360 0.006548 0.024844 0.052885 0.000656 0.045078 0.020934 1.000000 0.046377 27 / 57 30 / 58 29 / 60 43 / 93 160 / 502 207 / 685 117 / 325 81 / 223 78 / 209 94 / 323 83 / 230

GO:0008203 GO:0016126 GO:0022904 GO:0042981 GO:0060326 cholesterol metaboli... sterol biosynthetic ... respiratory electron... regulation of apopto... cell chemotaxis 1.000000 0.000138 0.000787 0.024194 0.040511 25 / 54 23 / 33 36 / 68 159 / 498 30 / 60

GO:0006695 cholesterol biosynth... 0.000171 22 / 31

GO:0003674 molecular_function 1.000000 1294 / 5591

GO:0003824 C catalytic activity 1.000000 522 / 2211

GO:0016491 oxidoreductase activ... 0.000597 103 / 287

GO:0016651 oxidoreductase activ... 0.003869 28 / 53

GO:0003954 GO:0016655 NADH dehydrogenase a... oxidoreductase activ... 0.032200 0.011438 19 / 33 22 / 39

GO:0050136 NADH dehydrogenase (... 0.032200 19 / 33

GO:0008137 NADH dehydrogenase (... 0.032200 19 / 33

Supplementary Figure S3 HIST1H3J 14 l COL15A1l

A 12

ARG2MS4A3

10 l l RAB3B l HIST1H2AB CYP26A1l ALDH1A1l l

HIST1H1B ZFHX3 BCL6 ACTL8 ODC1l l 8 HIST1H3H l l l l ALDOCl CREBRFl l MIR17HG

HIST1H2AH IRF2BP2ACSM3MIR3687ADAM19PYGL l ll l l l S1PR3 KIT l FAM178Bl l l EBNA1BP2 FN1 6 l CPZ KIAA1522TSC22D3PRG2 lC3 l ll TESK2l HIST2H3D RNF213GDF15TP53INP1ITGAM l POP1 ll HIST1H2BJ PLEKHH2l ll TNFRSF9 −log10 (FDR) l l HBZ MYCHIST1H2AI MIR3648DUSP10SEC24DFZD4l LIPH l ELF3 l ll LOC100506548SRRM2l SAl T1 SEMA3All l HIST1H2AL l l l l l l ACSF2 MIR663ABTG2 PNRC1LRP12JUNZNF467l PKLR ALDH16A1CD3EAPHBG1 ll ll l l MYLKl l l ENTPD1CYP3A5 SMIM3 l PAPPAl l DFNA5l TMEM51−AS1TUBB2Bl 4 l MARCKSL1 BTG1 PCSK9l l l lNR1D2PGLYRP4l l SLC38A5NOP56l LOC284801l GPRC5CSLAMF6HECW2l l l l l l l PPP1R15AlMXD1 ll EMILIN2l GBP2 CDCA7Ll l RABAl l C1BMF l SFXN2HBG2HIST1H4Kl ANKRD30BLPPP1R3BASS1LHFPL2l NCALDlPLAU INPP5JPROB1 l l l l HIST1H4I IRF2BPLSMARCA1LGALS1ll l lCHST3l l l BSN l lHIP1l lZP3 l l ll POLR3GSAPCD2NME1 RNASET2l l CCDC136DNAJB2l l SCARF2 IGFL2 l l ll ll SERPINB1l l l PALMl l CA12 l l TRIQKl EGFl FAM46B HIST1H4Jl ll NFl AT5 HELZ2l IRS2LRP1RNASE1lDHRS2l l l GDAP1 l l ll l l l ANAPC15 PHF21ADUSP13l CD84l l ll Fl AIM2 KISS1R RRP7Bl ZNF614l l ll l ZFP36L2NEAT1 l l CLIP2GLIS2 l TMEM177l l ZMIZ1DUSP4l lTLE2l l LBH l C19orf25l ll MANBAll MRC2FlAM1l SOGA2TEX1914A1ID2 l SHC2l l MANEALl l ll l llNFUSP35PPl ABBS2lARATC4l l l l LINC00200l l l l ST3GAL1lll l ll CSF1MIR3189l IL2RA TREML2l ERPNMTPVMER34−1l ASNORD97OSBP2QR4GCDHl l ll l ll l ANKRD33Bl ll EVl CSF3RA1BlLRRC4BCYP1A1lADTFEBAMTS14l CYP27A1 lZNF239l l l l l lll l l l llMARC2l l l l DBPl l l l l l l l l l l l ll l l l l l l l l l l l l l l 2 lll l l l l l l llllll l l ll ll l l l l l l l l l l l ll l lllll ll l l l l l l ll lll l l l l l llll l l l l ll l ll l l ll l l l l llll ll ll ll l l l l lll l l l l l l l l ll l l l llll lll l l l l l l lll l l l l l l l l l l l llll ll l l l l ll l l l lll ll l l l l l l l l l l l l l l l ll l ll l l l l l ll l lll l l l l l ll l l ll l l ll l l l l l l l l l l ll l ll l l l l l l lll l l l ll l l l lll ll l ll l l l lll l l l l l ll l l ll l lll l l ll lll ll lll ll l l l lllllll l lll l ll l l l ll ll l l l l l ll ll lll ll l lllll l ll ll l l l l ll l l l ll l ll ll l l l l lllllllll l lll l ll l l lll ll l lll l l l l l l l l l l l l l l l llllllllll l l l l ll l l l l l l l l l l l l l ll lllll lllllllllll l lllllll lll ll l lll lllll ll l l ll l l ll l l l ll ll lllll l lll lll lllll llll l lll lll lllllllll llll lll lllll l l ll ll ll l l l l l l l l l ll ll l l lll l llll l lllll l l ll l l l lll l l l l l l l lllll l lll l ll l l l l l l l l ll l l ll ll l l l ll ll llll l ll l l lll l l llll l l l l l l l l l l l l l l ll ll l llll llllllll llllllllllllllllllllll l lll l l lll l ll ll llll l l l l l l l l l l l l l l l ll llllllllll l l lllll l llllll llll lll ll lll l l l ll lll l l l l llll ll ll l l l lll l l l l l lll l l l l l l l l l l l lll ll l l lll l l ll ll llllll ll l l l l l l l l l l l ll lll l l l l l l lll l l l l l l l l l llllll llllllllllllllllll l l ll ll ll lll l llll ll l ll ll l l l l ll l ll ll l l ll lll l ll l ll l l l l l l l l l l l l llll ll l ll ll llll l ll l ll l l l l l l l l ll ll ll lll ll ll lll ll l l l l lllll l ll l ll l l ll l l l l l lll l ll lll l llll l l ll l ll l l l l l l l l l llll l lll l l l lll l l l l l l ll l l l l l l l l ll l l lll llllll llll lllllll lllllll l l lll llllllllllll lll l ll ll l l l l l l lllll ll l l l l l l l lllll l lll lll llll l lllllllllll l l l ll l lllllll llll l l l ll ll l ll ll l ll l l l l l l l l l ll l l l ll l ll l l l l l l l l l l l l ll ll l l l l l l ll l lll lllllllllllll lllllll l llllll l l l l l l lllll lllll ll l l llll llll lllll l ll llll l l ll l l l l l lll lllllll llllll l ll ll l ll ll l l l l l l l l l l l l l l l l l ll l l l l lll l l lll l l l l l ll l lll l l l l l l ll l l l ll l l l ll l l l lll ll ll l l l llllll lll l l ll ll l l l l l l l l l l l l l l l l l l ll l lll lllllll lll lll llll l l l l ll l l ll ll l l ll l l l l l l ll l l ll l l ll ll l ll l l l ll l ll l l ll l l l l ll l lll ll ll l l l lllll l ll l l l l ll l l l l ll l l lll l l l l l l l ll l l llll l lll lll llll llll llll ll lll ll ll l lll ll l l l l ll l l l l ll l ll ll l ll ll lll lllll l l l l llll l ll l l l l l l l l l l l ll l l lll l lll l ll l l l l l l l lll l ll l ll l l l l l l l l l lll lll l ll l l l ll l l l l l ll ll l ll lll ll lll ll lllll ll l l lll l l llll l llll l ll l l l ll l llll ll l l l ll l l l ll l l l l l l lll l l l l l ll l l ll ll lllll llll lll l l llllllll ll ll ll lll l llll l l l ll l l ll l l ll l l l ll lllllllllllllllll lll lllllllllllll llllll l l l ll lll lllllllll ll l l l l l l l l l ll ll ll l l l l l l l l l l l l l l l ll l l ll l l lllll lll ll l l l l llll lll l l l l l l l ll lll l l lll lll ll ll llllllllll ll l ll llllllllllllll lllllllll l l lllll lllll llll l llll lll lll ll ll l ll l l l l llll l ll l l lll l l l l ll ll l l l l l l lll ll lll l l l l ll l l llll llllll l ll l l l llll l l l l l l l l l l ll l lll l l l l l ll ll ll ll l l l ll l l ll l lll l l l lll l l l l l l l l l l ll llllll l lll l l l l l l l l l l l l l l ll ll l l l l l l ll l ll l l l l l l l l l l l l l lllllllllll llllllllllllllllllllll lllllll ll ll llllll llllllllllllllll lllll lllllll l lll ll llll llll l ll l l ll l l ll l l l l l l l l l l llll l lll l llll ll ll lllll ll l lllllll ll ll l ll l ll l l l l l l l l l l l l l l l l lll l lll lll l ll ll l l l llll lll l l l llll l l l l l l l l l l l l l l l l l ll l l ll ll l l l l llll l ll ll l lll l l l ll l l l l l ll l l l l l l l lll lllll l ll l ll l l l l l l l l l l l l l l l l l l l l l ll l ll ll l l l l ll l l ll l ll l l ll l ll l ll ll llll l l l l ll l lll l l ll l ll l l ll l ll l l l llllllll l lll l ll llll ll lll l l lll l l l l ll lllllllll l l lll l ll ll ll l l l l ll l l l l ll l l l ll l l l l l l l l l l llllll l lll ll ll l l llll l l lll l ll ll llll ll llll l l l l ll ll l l l l l l l l l ll ll l l l l l l l l l l l l lll l l llll l lll lll ll ll l l ll l ll llll l l ll l l l l l l l l ll l lllll llll lllll ll l llllllll l lllllll lll ll l llll l l ll l l ll l ll l l l ll l l l l l l lllll ll lll ll lll l l ll l l l lll l ll ll l l l l l ll l l l l l l l l l ll l l l l l l l l l lll lll l llllll l ll l l l l l l l l l l l ll l l l ll lllll ll lll l ll l lll ll llll lllll l ll l l l l l l l l l ll l l l l l l l l l ll lll llll lllllllllll lllll l l llll lll l lllll lll l l l l l l l l l l l l l llllllll l l lll l l l l l l l l l l l l ll llllllll l l l ll l l l l lllllllll l l l lll l lll l l l ll l l l lllll ll lll l l ll l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l ll l l l l l ll ll l ll l l lllll lll ll ll ll llllllllllllllllllllll llllllll l l l lllllllllllllll lllll llllllll l llll l l ll ll llll l l l l l l l l l l l l l l lll ll llll l llll l l ll l l l ll ll l ll l llll l l l l lll ll l l l l l l l ll l l l l llll ll l ll l ll l l ll l l l l l l l l l l l ll l l llll l ll l l lll l l l llll ll l lll l l l l l l l l l l l l l l l l l ll ll l l ll lll l ll lll lllll l ll l lll l l l l l l ll l l l l l l ll l ll l l l l l l l l l l ll ll l l l l l llllll ll l l lllll lll ll l l ll l l l l l lllll l llll lll l l l l l l l ll l l l ll l l ll lll l ll ll lllll l lll lll l l l lll lll l ll l l l l l l l l l l l llll lllllllllllllllll l llllllll l l ll ll ll ll lll llllll lll llllll ll l llll l l l l l l l l l l ll llll l l l l l l l ll l l l l l l ll l ll lll l llll llllll l ll llllll lll lllll l l ll l l l l l l l l ll l l l l l l l l l l l ll l l l l ll ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l llll l l llllllll llll lll l ll l l llll lll l l ll l ll ll lll l l l l l l lll lll lllllllllll l ll ll l l ll ll l l lll l ll ll l l l l l l l l l l llll l l l l l l ll l l l l l l l l l ll ll l lll lll llllllllll llllllllllll llll l ll lll l ll lll lll l lllllll lllllllllll ll lll ll l l ll llllll lll ll l l l ll lll l l l l l l l ll l l l l ll l l l l lll l ll lll l l l l l l l l l l l l l l ll lll ll lllll llllllllllll lllll l llll l l lll ll l llll l l l l l lll l l l l l l ll l l l l l l l l ll l l l l ll llll lll ll lll l ll l l lll llll l l l l ll l l lllllllll l ll l l llll l l l l l ll l l l l l l l l l ll llll ll ll l l ll llllll ll lllll ll ll lll ll l l ll lllll l l llllllll llllll l l l l l ll l l lll l l l l l l l l l l l l ll l l l llll l l ll l l l l lll l l lll l l ll ll l l l l ll ll ll l l l l l ll l ll lll l lll l ll ll ll l l l l l l l l l l l l l ll ll l l l l l l l lll lll l lll lllll ll ll llll lll l l llll l lll l l l ll l l llllllll l l lll l ll l l ll l l l l l l l l l lll l l l l llllll ll lll lll l l l l l l l l l l l l l l l l l l l ll l l l l l lll ll l l l llll lll l l ll l ll l ll ll l l l l l lll llllll ll l llll ll lll l l lllll l l l l l l l l l lll l l l l llll ll l llll lll l l l l l l l ll l ll l l lll lllll ll ll l ll l l l lllll l l l l lll l l l l l l l l l l l l ll ll l l l l l l ll l l l l l l l l l ll l l l l l l l l l l ll l l ll lllllll l l l l ll l l l l l l l l l l l ll l ll ll l l l l l l l l l ll l l l l l l l ll ll ll ll lllllll llllllll lllll llllllll ll ll ll l llllllll llllllll llllllll ll lll lllll lllll l l l ll ll l l l l l l l l l l l l l l l l l l l ll l l ll ll l l l l l l l l l l l l l l l ll l lll l l l l l lllllll l ll l l ll ll ll l l ll l l l ll l l l l l ll l l l l l l l l l ll l ll l l l ll ll ll ll l l l l l l ll l l l l ll llll lllll l l l l llll lllllllllll l l l l l l l l l l l l l l ll lll ll ll l ll llllllllll l lll l lll l lll l l ll ll l l ll ll lll lll lll lll ll ll l l l l ll l ll l l l lll ll l ll l l l ll l l ll l l l l l l l l ll l l l l l l l l ll l l lllll l l lll lll l llllll ll l l l ll ll lllll l ll l l lllll lll l l ll l l l lll l ll ll ll l l l lllll llllllllll lll llll l l l ll ll llllllllll l ll l l ll llll ll l l ll l l ll ll l l l l l l l l l ll l l l l l l ll lll ll lllll ll l l l l l ll l l ll l lll l l l l l ll l l l l ll ll l llll l l l ll l l l l ll l ll l l l ll lll l ll l l lllll ll l l ll l lll l l l l ll ll lllll l l l ll lll l l l l lll l l l ll lll ll l l l l l l l ll ll ll l l ll l l l ll ll l ll l l l l l l l l l l l l l l l l l l ll ll llll lll ll l l lll l l l ll ll l l l l ll l ll l l ll llllll llll l ll ll l ll lllllll l l l l l l l ll l l l ll ll l l l l l ll lll ll ll ll l l l l l l l l l l l ll ll ll l ll lll l l l l l llll ll l l ll l l l l l l l l l l l ll ll l l ll ll l lll lll lll l l l l lll ll lllll l ll l l l l l l ll l l l l l l l l l ll ll l l ll l l l l l l l l l l l l ll lllll l l l l l l lll l l l ll l l l l l l l l l ll ll l l ll l ll l ll l llll l l l l l ll l l l l ll l l l ll ll l l l ll l l l l l l l l l l l l l l l l l l ll l ll l ll l l ll ll l l l l l l l l l l l l l l ll lll ll l lll ll ll ll l ll l l l l l l ll l l l l l l l l l l l l l l l l l l l l llll lll ll l l l ll l lll l l l l l ll l l l l l ll l l l l l l l l l l l l l ll l l l l l l l ll l l l l l l l l l l l l l l l ll l l l l l l l l l l lll l lll l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l ll ll ll ll l l l l lll ll l l l l l l l l l l l l l l ll l l l l l l l ll l l l l l ll ll ll llllllll lllllllllll l l l l l lllll ll l lllll lll ll ll l ll l l l l ll l l l l l l l l l llllllllllllll lllllll llll lll l ll llll llll l ll l ll llll l l l ll l l l l l l ll l l llll l l l l l l ll l l lll l ll l l l l l l l llll l l l l ll ll l l l l ll l l l l l l ll l l lll l l l l l ll l l l l l l l l l l ll l l l l l ll ll l l l l ll l l l l lllll ll l ll lll l l l l ll ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l lllll l ll ll ll l lllll llll l l l ll l l l l l l l l l l l l l ll llll l l ll l lllll ll ll l l l l l l l lll l lllllll lllllll l l l llll lll lll lll ll l l l l l l l l l ll l l l l l l l l l l l l l l l ll l l l ll ll lll l ll l l l llll l l l l l l ll l l l l l l l l l l l l ll lll l l l ll l ll l l l l l ll ll ll l l l l l ll l ll llll l ll l l llllll l ll l ll l l l l l l l l ll l l l ll l l l l l l l l l l l l l l l l ll l l l l ll l l l llllllll llllllll lllll llll l llll llll ll ll llll lllll llllll l l ll l l ll l l ll lll ll l l l l l lll l l l l ll l l l l l l l l l l l l ll l ll l lll ll ll ll ll llllll l lllll ll l lll ll lllll lll ll ll ll l l l ll l l l l l l l l l l l l ll l l l l l l l l l l l lll ll ll lll ll l lll l lll llll l l ll l l l l l ll l l l l ll l l l l l l ll ll l llll llllllll l l ll lll l lll ll l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l ll l l l l l l l l lll ll ll l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l lll ll l l l l ll l l ll ll l lll llll lll l l l l ll l l l l l l l l l l ll l l l l l l l l l l l l l l ll l ll l l ll l lll lll l l ll ll l l ll l l l l l l l l l l l l l l ll l l ll l ll l l l ll ll ll lll ll lll l l l l l l l l l l l l ll l ll l ll l l l ll l lll l ll l l l l l l l l l l ll l lll lllll l l lll llllllllll lll lll lll ll llll lllll llll l llllllll ll ll l ll l l l l ll l l l l llll l ll ll l l l l ll l l l l l l l l l l ll l l ll l ll l l l l l l l llll l l l l l l l l l l l l l l l l l l l l ll lll l l l l l l l l ll l l l l lll l lllll l ll ll lll llll ll lllll l l l l l l l l l l l l ll ll l l l l l l l l l l l l l ll lll ll l l l l l l l l l l l l l l l l l l l ll l l ll l l l ll l ll l l l lll llllll l ll ll l l l l l l lll l l l ll l l l ll l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l lll l l l l lll ll llll l l ll lll l l ll ll l l ll l l l l l ll l l l l l l l l ll ll l ll l l l l l ll l ll l l l l l l l l l l ll ll l lll l l l l l l l l l ll l lll ll l l l l l l l l l l l l l l l l l l ll l ll l ll ll l l lll ll llll ll l l l l lll l ll ll l l l l ll l l l ll l l l l l l l ll l l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l lll l l l l l l l l l l l l l l l ll l l l l l l l l ll l l l l l l l ll l l l l l l l l l l l l ll l lll l l l l l l l l l lll l llll l l l l l l lllllll l l ll l l l l l l l l l l l l l l l l l ll l lll lll l l l l l l ll l ll l l l lll l l l l l ll l lll l l ll ll l l llll l ll l l lll l ll ll lllll l ll ll l l lll l l l l ll ll l l ll l l l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l llll l l llllllllllllll ll lllllll l lll llll llll l llll lll l l ll l l l l l l l l l l l l l l l l l l ll l l ll l l l l l l l lll ll lll l l ll ll l l lll ll l l l l l l l l l llllllll lll l l l l llllll lllll ll llll llll l ll lll l ll l l l l l l l l l l l ll l l llll l l ll ll l l l l l l l l l ll ll l l ll l l l lll l ll l llll ll ll l lll l ll lll l l l l l l l l l l l l l l l l l l l l l l l l l ll l l l l l l ll l l l l l l l l l ll l l l l l l l l l l l l l l l l l ll l l ll ll ll l l l l l l l l l l lll ll l ll ll l l l l l l l ll l l ll l ll ll l ll ll l l ll l l l l l l l ll l l l ll ll lll l l l l l l ll l l ll ll l l l l l llllllll l ll l l l l ll l l l l l l l l l l l l ll l l ll ll l l lll l l l l l l l l l l ll l l l l l l l l l l l l l l l ll l lll ll l l l ll lll l l l l l l l l l l lll l l l l ll l l l l l l l ll l l l l l l l l l l lll l l l l l l l ll l l l l llll l l l l l l l l l l l l l l l l ll ll l lll l l ll l l l l l l l l l ll l l lll l l ll l l l l l l l l l l l l l l l l l l ll l l l lll l ll l lll l l l l l ll l l l l l l l lllll l llll l l lllll l l l l l l l l l l l ll l l l l l l l l l l l l l ll lll l l lll l l l l l l l l l l l l l l l ll l ll l l ll l l l l l l l l l l l ll ll l l l l l l l l l l l l l ll l l l l l l l l l l l ll lll l lll l l l ll l l l l l ll l l l l l l ll l lll l l l l l ll ll l l ll l ll l l ll ll l l l l l ll l l l ll ll ll l l l l l lll l l l l l l l l l l l l l ll l l l l l l l l l l l l ll l l ll l l l l l l l l l ll llll lll l l l l l l l ll ll l l l ll l l lll l ll ll l l l l l l l l l l ll l l l l l l l l l lll ll l l l l l l l ll l l l l l l l l l l lllll ll l ll l l l l l l l ll l l l ll llll llll l ll l l l l l l ll l l ll ll llll lll llll l l l l l l ll l l l ll l l l l l l l l lll l l l l l l ll l l l lll l l l l ll ll l l l ll ll l l l l l l l ll ll l lll l l ll l l l l l l l l l l l l l ll lll l l ll l ll ll l l l l l l l l l ll l l ll l l l ll l l l l ll ll l l l l l l l l l l ll llll l ll l l l l lll l l l l l l l l l l l l l l l l ll l l ll l l lll lll l lllll ll ll lll l l ll l l l l l l l l l l l ll l ll l l l l l l l l l ll lll l l l ll l l ll l l l l l l l llll l l l l ll l lllll l llll l l l l l l l l l l l l ll l l l l l l l l l l l l l l llll llll ll lll ll ll l ll lll l l l l l l l ll l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l lll l l l l l l ll l l l l l ll ll l ll ll l l l l l l l l ll l l ll ll ll l l l l l l l l l l l l lll l l l l l ll l l l l l l l l ll l l l l l l l l ll l lll llll l l l ll l l l l l l l ll l l l l l l ll l ll ll l ll l l l ll l ll l l l l ll llll ll l l l l l l lll l ll l l l l ll ll l l ll l l l l l l l l lll ll l ll ll ll l l lll ll l lll llll l l l l l l l ll l l ll ll l l l l l l lll l l lll ll l lll l ll l lll ll l l l l l l ll l l ll l l ll l l l l l l l l l l l l l l l l ll l l l l l l l l l l l l l l ll ll l l l l l l l l l ll l l l l l l l ll l l l l lll llllllll l l ll ll l lll l l l l l l ll llll l l l l l l lll l l l l l l l l l l l l l ll l l l ll l l l ll lll lll llllll ll l ll ll lll l l l l l l l l ll l ll l l l l l l l l l l l l ll l l l llll lll l l l l lll l lll l ll l l l l l l l l l l l l l l l l l l l l l ll l l l l l l ll ll l l l l ll l l lll ll ll l ll l l l l l lll l llll l lllllll lllll l l ll l l ll l ll l l l l l ll l ll ll l l l l l l l l l l l l lll ll l l lll l l l l l l l l ll l l l l l l ll l l ll l l lll l ll lll l l lll l ll l l l l ll l ll l l l l llll l l l l l l l l l ll l l l l l l ll l lll l l l llll l l l ll l l l l llll l ll l l l l l l l l l l l l lll l ll ll ll l l ll l l l l l lll ll lll l l lll ll ll l l l l l l ll l ll l ll lll lll ll l lllll l llll l ll ll l l l l l llll ll l lll l l l l l l l lll l l l ll l l l ll l l l l l ll l lll ll l ll l l l l l l l l l l ll lll l llll ll ll l l l l l l l l l l l l l l l llllll l l ll l l ll l l l lll l lll l l l l l ll l ll l l l l l l l l l ll l l ll lllll l l l lll l lll l ll ll l ll l l ll l l l l l l l l l l ll l l l ll ll l l lll ll l l l l l l l l ll l l ll l llll l l l l l l l l ll l l l l ll l l l ll l l ll l l l l l l l l l l l l ll l l lll l l l l l l l lll l l ll l ll l ll l l l l l lllll llllll lll lllllllllllllll l l l l ll l l l l ll l ll l l l l l l l ll l l l l l l lll lll lll l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l lll l l l l l l l l l l l l ll l l l l ll l ll ll l llll l l l l ll l l l l l l l ll l l l l ll l l ll l l l l l l l l l ll llllll ll ll lll l l l lll l ll l l l l l l l l ll l ll ll l l l l l l l ll l ll ll ll lll l l l l lll l ll ll l l l l l ll l ll l l l l l l l l l l ll l l l l l ll ll l l l ll l l l l l l l l l l l l l l l l l l ll l l lll l l l ll l l l l llll l l l l l l ll l l l l l ll llll l l l l l l l llll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l lll l l lllll l l l l l l l l l l l l l l l l l l lll l ll l l l l l l l l l l l l l l l l l ll ll ll l l ll l l ll ll lll l lll l lllll l ll ll l llll lllllllllll llllll lll l ll l ll l l l l l l l l l l l l l l l l l ll l l l l l l l l l l l ll l l l l l l l l l l l l l l l l l l l l l l ll l ll l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l 0

−2 −1 0 1 2 3

log2 (SK053 / Control)

regulation of megakaryocyte differentiationnegative regulation of megakaryocyte differentiation

negative regulation of myeloid cell regulation of myeloid cell differentiation differentiation regulation of immune system process

regulation of multicellular organismal B process positive regulation of developmental process negative regulation of cell differentiation positive regulation of biological process regulation of developmental process regulation of cell differentiation

regulation of biological process negative regulation of developmental process negative regulation of biological process response to bacterium positive regulation of cell differentiation response to biotic stimulus response to other organism regulation of cellular processnegative regulation of cellular process positive regulation of cellular process defense response to bacterium

biological regulation regulation of muscle cell differentiation multi-organism process

regulation of cellpositive proliferation regulation of muscle cell differentiation defense response response to stimulus response to stress positive regulation of cell proliferation

biological_process

metabolic process

small molecule metabolic process cellular component biogenesis

cellular component organization cellular process

macromolecular complex subunit DNA conformation change organization cellular component assembly

DNA packaging cellular macromolecular complex organelle organizationsubunit organization

macromolecular complex assembly

chromatin assembly cellular macromolecular complex assembly

nucleosome organization chromosome organization chromatin assembly or disassembly

protein-DNA complex assembly nucleosome assembly C chromatin organization

metal ion binding

cation binding

transition metal ion binding

heme binding oxygen binding

ion binding

tetrapyrrole binding iron ion binding

binding

molecular_function Supplementary Figure S4 Ctrl 1 h 4 h 24 h 48 h 72 h 96 h 120 h

BiP

P-eIF2a

XBP1

GAPDH

Supplementary Figure S5 A B Growth of HL60 cells HL-60 MOLM14

10 NTC TXN(b) TXN(c) NTC TXN(b) TXN(c) controls 8 NTC WB:TXN

] TXN(a) 6 6 4 [x 10 2 WB:GAPDH 0 0 2 4 6 8 10 Number of viable cells time of knockdown [d]

C HL-60 D MOLM14

NTC NTC

* * * * * * TXN(b) * TXN(b) **

* *

Days of culture Days of culture * TXN(c) * TXN(c) ** * 0 5 10 15 20 25 0 2 4 6 8 Number of viable cells [x 106] Number of viable cells [x 106] E 50

40 HL-60 MOLM14 cells + 30 1b p=0.0005 20 p=0.032 p=0.044

% of CD1 10 NS

0 TXN TXN TXN TXN NTC (b) (c) NTC (b) (c)

Supplementary Figure S6 A B CEBPA NTC PDI(d) NTC (a) (c) 1 2 1 2

C/EBPα C/EBPα 42 kDa 42 kDa C/EBPα C/EBPα 30 kDa 30 kDa

GAPDH GAPDH

C SK053 CEBPA(a) CEBPA(c) 0 µM 0 µM 5 µM 5 µM

] 10

6 10 µM 10 µM

Number of viable cells [x 10 0

Days of culture D

40 SK053 0 µM

cells 30

+ 5 µM

1b 10 µM 20

10 % of CD1

0 NTC CEBPA(a) CEBPA(c)

Supplementary Figure S7 A

CF3 O O R O N SK053 H CF 3

B SK053-biotin = SK-BIO

C inactive biotinylated SK053 analog = SK-IN

CF3 O O H O N O N O H O CF3

leaving group truncated SK053

O O

N R N R SH + SK053 S + SK053 S H H

tioredoksynaPDPIDI tioredoksynaPDPIDI tioredoksynaPDPIDI H SH S SH N R

+ O CF O 3 + OH

CF CF3 O 3 OH

CF 3

Supplementary Figure S8 A 1 MKHHHHHHPM SDYDIPTTEN LYFQGAMASV PDAPEEEDHV LVLRKSNFAE 51 ALAAHKYLLV EFYAPWCGHC KALAPEYAKA AGKLKAEGSE IRLAKVDATE 101 ESDLAQQYGV RGYPTIKFFR NGDTASPKEY TAGREADDIV NWLKKRTGPA 151 ATTLPDGAAA ESLVESSEVA VIGFFKDVES DSAKQFLQAA EAIDDIPFGI 201 TSNSDVFSKY QLDKDGVVLF KKFDEGRNNF EGEVTKENLL DFIKHNQLPL 251 VIEFTEQTAP KIFGGEIKTH ILLFLPKSVS DYDGKLSNFK TAAESFKGKI 301 LFIFIDSDHT DNQRILEFFG LKKEECPAVR LITLEEEMTK YKPESEELTA 351 ERITEFCHRF LEGKIKPHLM SQELPEDWDK QPVKVLVGKN FEDVAFDEKK 401 NVFVEFYAPW CGHCKQLAPI WDKLGETYKD HENIVIAKMD STANEVEAVK 451 VHSFPTLKFF PASADRTVID YNGERTLDGF KKFLESGGQD GAGDDDDLED 501 LEEAEEPDME EDDDQKAVKD EL

# b b++ b* b*++ b0 b0++ Seq. y y++ y* y*++ y0 y0++ # C 1 129.1022 65.0548 112.0757 56.5415 K 16 2 243.1452 122.0762 226.1186 113.5629 N 2081.9612 1041.4842 2064.9347 1032.9710 2063.9506 1032.4790 15 3 342.2136 171.6104 325.1870 163.0972 V 1967.9183 984.4628 1950.8917 975.9495 1949.9077 975.4575 14 4 489.2820 245.1446 472.2554 236.6314 F 1868.8499 934.9286 1851.8233 926.4153 1850.8393 925.9233 13 5 588.3504 294.6788 571.3239 286.1656 V 1721.7814 861.3944 1704.7549 852.8811 1703.7709 852.3891 12 6 717.3930 359.2001 700.3665 350.6869 699.3824 350.1949 E 1622.7130 811.8602 1605.6865 803.3469 1604.7025 802.8549 11 7 864.4614 432.7343 847.4349 424.2211 846.4508 423.7291 F 1493.6704 747.3389 1476.6439 738.8256 10 8 1027.5247 514.2660 1010.4982 505.7527 1009.5142 505.2607 Y 1346.6020 673.8047 1329.5755 665.2914 9 9 1098.5619 549.7846 1081.5353 541.2713 1080.5513 540.7793 A 1183.5387 592.2730 1166.5121 583.7597 8 10 1195.6146 598.3109 1178.5881 589.7977 1177.6041 589.3057 P 1112.5016 556.7544 1095.4750 548.2412 7 11 1381.6939 691.3506 1364.6674 682.8373 1363.6834 682.3453 W 1015.4488 508.2280 998.4223 499.7148 6 12 1484.7031 742.8552 1467.6766 734.3419 1466.6926 733.8499 C# 829.3695 415.1884 812.3430 406.6751 5 13 1541.7246 771.3659 1524.6980 762.8527 1523.7140 762.3606 G 726.3603 363.6838 709.3338 355.1705 4 14 1678.7835 839.8954 1661.7569 831.3821 1660.7729 830.8901 H 669.3389 335.1731 652.3123 326.6598 3 15 2063.9506 1032.4790 2046.9241 1023.9657 2045.9401 1023.4737 C* 532.2799 266.6436 515.2534 258.1303 2 16 K 147.1128 74.0600 130.0863 65.5468 1

D Score Mr(calc) Delta Sequence Site Analysis 56.0 2209.0489 0.0059 KNVFVEFYAPWCGHCK SK053 C15* 87.57% 47.5 2209.0489 0.0059 KNVFVEFYAPWCGHCK SK053 C12# 12.43%

E PDIA1_d4 PDIA1_d4 + SK053 (1:10)

13396.5010 13960.5010

Supplementary Figure S9 A

Supplementary Figure S10