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Figure S1-S6, Table S1 Supplementary Figure Legends Supplementary Figure S1. CETSA assay showing on target binding of JQ1: CHLA-10 and CADO-ES1 cells were treated with DMSO or 500nM JQ1 for 3h and incubated at indicated temperatures for CETSA (cellular thermal shift assay). Cells w ere lysed and soluble proteins detected by immunoblotting with the indicated antibody. Shown are representative blots from two independent experiments. b) EWS/ERG and BRD4 interaction is acetylation independent. Top-panel, CADO-ES1 cells were treated with DM SO or HDAC inhibitors (TSA and SAHA) for 48h followed by nuclear extractions. Immunoprecipitation followed by immunoblotting for the indicated target was performed. IgG was used as negative control. Lower panel, HDAC treatment control. Increased pan-acetylated histone levels in TSA and SAHA treated nuclear lysates compared to DMSO control. c) Endogenous association of EWS/FLI1 and BRD4. CHLA-10 nuclear extracts were subjected to immunoprecipitation using anti-FLI1 antibody. Immunoprecipitates were analyzed for the presence of BRD4 by immunoblotting. Supplementary Figure S2. a) Table showing high-throughput sequencing read information for RNA-seq libraries of indicated samples performed for this study. b) Lack of MYC downregulation upon knockdown of BRD2, BRD3 or BRD4. qRT-PCR showing the GAPDH normalized expression of MYC in the indicated cells. c) RT-qPCR validation of PHF19 downregulation upon JQ1 treatment in ES cells. d) RNAseq FPKM values for FLI1 and ERG in the indicated cell lines treated with DMSO or 500nM JQ1 for 48hrs. Supplementary Figure S3. PHF19 is the only gene affected transcriptionally by EWS/FLI1 or BRD4 knockdown or JQ1 treatment in the chromosome 9 loci that include genes PSMD5, FBXW2, MEGF9 and TRAF1 flanking PHF19. a) Genome browser view of H3K27ac and EWS/FLI1 at PHF19 loci in SK-NM-C and A675 cells. The ChIP-seq data were downloaded from NCBI GEO with accession number GSE619534. b) Normalized FPKM values for PHF19, PSMD5, FBXW2, MEGF9 and TRAF1 transcripts in the SK-N-MC and A673 cells expressing control shRNA or shFLI1 (GSE61953). For CHLA-10, data for shBRD4 is also included. c) As in b, for CHLA10, RD-ES, SK-N-MC and CADO-ES1 control versus JQ1 treatment. Supplementary Figure S4. ChIP with the indicated antibodies using chromatin extracted from DMSO or 0.5uM JQ1 for 24h. qPCR was performed using a primer set amplifying a region flanking the EWS/FLI1 binding site located approximately 20kb from the PHF19 gene (hg19 - chr9:123,636,835-123,636,855). Fold enrichment relative to the IgG control from DMSO is plotted with standard deviation. b) qRT-PCR and immunoblot validation of PHF19 knockdown in SK-N-MC cells transfected with 50nM si-nontarget or siPHF19 for 48hrs. c) cells as in b were treated with or without 500nM JQ1. Cells were counted every alternate day for 6 days. Inset shows zoomed growth curves for JQ1 and siPHF19 + JQ1 treated cells. d) Increased sensitivity to JQ1 in PHF19 knockout cells. Cell viability curves for control or PHF19 knockout SK- N-MC cells treated with JQ1 for 4-days followed by cell-titer glow assay for viability. Mean ± s.e.m from n = 4 is plotted. Supplementary Figure S5. PHF19 overexpression correlates with overall survival in Ewing sarcoma patients. Kaplan-Meier analysis for PHF19 expression under the condition of overall survival probability in Ewing sarcoma patients using SurvExpress database. HR denotes hazard ratio. Insert, boxplot for the mRNA levels of PHF19 in high (red) and low (green)-risk cohorts. Supplementary Figure S6. a) Individual tumor volume for all three Ewing sarcoma xenograft models treated with vehicle or 50mg/kg JQ1 for 5days/week. b) Animal weights for tumor growth studies displayed as mean ±SEM. c) qRT-PCR for PHF19 and MYC in the CHLA-10 tumors from control and JQ1 treated mice. N =5 tumors from each group. d) Immunoblot analysis of PHF19 and MYC in CHLA-10 tumors as in c. GAPDH was used as loading control. e) Normalized quantification of PHF19 and MYC from d. P – values determined by Student’s t-test using GraphPad Prism. Supplementary Figure S1 a CADO-ES-1 cells DMSO JQ1 (500nM) 39 42 45 48 51 54 57 60 39 42 45 48 51 54 57 60 Temp (° C) BRD4 GAPDH CHLA-10 cells DMSO JQ1 (500nM) 39 42 45 48 51 54 57 60 39 42 45 48 51 54 57 60 Temp (° C) BRD4 GAPDH b IP-BRD4 c Input IgG IP-FLI1 Input IgG DMSO TSA SAHA BRD4 EWS/ERG EWS/FLI1 * IgG Heavy * IgG Heavy BRD4 DMSO TSA SAHA pan-acetylated histone Total H3 Supplementary Figure 2 a # Sample ID Reads Number Of % Total Reads Aligned 1 CHLA10-DMSO-1 151/151 13,213,552 94.45% 2 CHLA10-DMSO-2 151/151 10,756,525 93.40% 3 CHLA10-shFL1-1 151/151 35,988,604 93.36% 4 CHLA10-shFLI1-2 151/151 38,358,307 95.10% 5 CHLA10-shBRD4-1 151/151 39,119,332 94.33% 6 CHLA10-shBRD4-2 151/151 72,529,289 94.37% 7 CHLA10-JQ1-1 151/151 25,263,070 94.72% 8 CHLA10-JQ1-2 151/151 17,252,188 93.42% 9 RD-ES-DMSO-1 151/151 34,920,613 91.87% 10 RD-ES-DMSO-2 151/151 27,102,776 93.09% 11 RD-ES-JQ1-1 151/151 34,041,127 91.04% 12 RD-ES-JQ1-2 151/151 49,410,157 90.94% 13 Cado-ES-DMSO-1 151/151 11,777,772 91.50% 14 Cado-ES-DMSO-2 151/151 37,251,074 91.36% 15 Cado-ES-JQ1-1 151/151 15,570,290 91.23% 16 Cado-ES-JQ1-2 151/151 34,188,934 91.08% 17 SK-N-MC -DMSO-1 75/75 18,053,003 94.28% 18 SK-N-MC -DMSO-2 75/75 18,720,436 93.81% 19 SK-N-MC -JQ1-1 75/75 19,668,526 93.88% 20 SK-N-MC -JQ1-2 75/75 14,486,565 93.62% 21 SK-N-MC -PHF19_KO-1 75/75 84,317,814 92.83% 22 SK-N-MC -PHF19_KO-2 75/75 23,938,928 92.65% b CHLA-10 RD-ES CADO-ES1 c CHLA-10 RD-ES SK-N-MC CADO-ES1 2 1.2 1.2 1.2 1.2 1.4 1.2 1.0 1.0 1 1 1.2 1.5 1 0.8 0.8 0.8 1 0.6 0.6 0.6 0.4 0.4 0.4 0.5 0.2 0.2 0.2 Relative MYC mRNA 0 0 0 0 0 0 0 Relative PHF19 mRNA JQ1 JQ1 JQ1 JQ1 DMSO DMSO sh NT sh NT sh NT DMSO DMSO sh BRD2sh BRD3sh BRD4 sh BRD2sh BRD3sh BRD4 sh BRD2sh BRD3sh BRD4 d CHLA-10 RD-ES SK-N-MC CADO-ES1 Normalized FPKM (EWS-FLI1) (EWS-FLI1) (EWS-FLI1) (EWS-ERG) Supplementary Figure 3 a Scale 100kb hg19 Chr9: 123,500,000 123,550,000 123,600,000 123,650,000 123,700,000 _ A673 FLI1 _ SK-N-MC FLI1 _ A673 H3K27ac _ SK-N-MC H3K27ac PSMD5 MEGF9 FBXW2 PHF19 TRAF1 b SK-N-MC A673 CHLA-10 dmso sh Ct. 50 35 30 sh Ct. sh FLI1 sh FLI1 45 sh FLI1 30 sh BRD4 25 40 35 25 20 30 20 15 25 20 15 10 15 10 10 Normalized FPKM 5 5 5 0 0 0 PHF19 PSMD5 FBXW2 MEGF9 TRAF1 PHF19 PSMD5 FBXW2 MEGF9 TRAF1 PHF19 PSMD5 FBXW2 MEGF9 TRAF1 c CHLA-10 RD-ES SK-N-MC CADO-ES1 dmso dmso dmso dmso JQ1 JQ1 JQ1 JQ1 Normalized FPKM PHF19 PSMD5 FBXW2 MEGF9 TRAF1 PHF19 PSMD5 FBXW2 MEGF9 TRAF1 PHF19 PSMD5 FBXW2 MEGF9 TRAF1 PHF19 PSMD5 FBXW2 MEGF9 TRAF1 Supplementary Figure 4 a ChIP qPCR * P < 0.05 24 FLI1 ab BRD4 ab 20 IgG 16 12 n.s. Fold enrichment 8 4 n.s. 0 JQ1 JQ1 JQ1 DMSO DMSO DMSO b c si-NT si-PHF19 600 JQ1 PHF19 10000 siNT siPHF19 + JQ1 500 siPHF19 GAPDH JQ1 400 1.2 siPHF19 + JQ1 300 * P < 0.05 1 1000 * P < 0.0001 200 * P < 0.05 0.8 100 Percentage Cell Number * P < 0.05 * P < 0.05 0 0.6 * P < 0.05 0 2 4 6 100 * P < 0.05 0.4 Days 0.2 Percentage Cell Number Fold phf19 mRNA expression Fold phf19 mRNA 0 10 si-NT si-PHF19 0 2 4 6 Days d Control 1 00 PHF19 KO y t i l i * b a i v * l 5 0 l e C * * p < 0.001 * 0 * 0 .001 0 .01 0 .1 1 1 0 J Q1 (µM ) Supplementary Figure S5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 PHF19 mRNA expression PHF19 mRNA 4.5 Low risk High risk Time (months) Supplementary Figure S6 a SK-N-MC b SK-N-MC 30 2500 Vehicle treatment 2500 JQ1 treatment ) ) 3 3 2000 2000 (grams) 20 t 1500 1500 vehicle (n = 8) weigh 10 1000 1000 al JQ1 (n = 12) m ni 500 500 A Tumor Volume (mm Volume Tumor 0 Tumor Volume (mm Volume Tumor 0 0 1 4 7 9 11 14 16 18 21 22 1 4 7 9 11 14 16 18 21 22 1 4 7 9 11 14 16 18 21 22 Days of drug treatment Days Days CHLA-10 CHLA-10 Vehicle treatment JQ1 treatment 3000 3000 30 ) 3 ) 3 2500 2500 2000 2000 (grams) 20 t 1500 1500 vehicle (n = 8) weigh 1000 1000 10 al m JQ1 (n = 8) 500 500 ni Tumor Volume (mm Volume Tumor A Tumor Volume (mm Volume Tumor 0 0 0 1 4 7 9 11 14 16 18 22 1 4 7 9 11 14 16 18 22 1 4 7 9 11 14 16 18 22 Days Days Days of drug treatment CADO-ES1 CADO-ES1 2000 Vehicle treatment 2000 JQ1 treatment 30 ) 3 ) 3 1500 1500 20 (grams) t 1000 1000 vehicle (n = 10) weigh 10 al 500 m JQ1 (n = 10) 500 ni Tumor Volume (mm Volume Tumor A Tumor Volume (mm Volume Tumor 0 0 0 1 4 6 8 11 14 18 1 4 8 11 14 16 18 1 4 8 11 14 16 18 Days of drug treatment Days Days Vehicle tumors (n = 5) Vehicle tumors (n = 5) c JQ1 tumors (n = 5) d e JQ1 tumors (n = 5) 1.2 * p <0.0001 n.s.
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