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Supplemental Figures Supplemental Figures Supplementary Fig 1. External validation of GSTO1 with patient survival and relation of GSTO1 expression to tumor stages and grades. A, External validation of GSTO1 with patient survival using Rembrandt and Gravendeel Glioma data sets. B, GSTO1 expression is higher in GBM compared to LGG in TCGA data sets. C, GSTO1 expression increases in KIRC is significantly different between tumor stages and grades. Supplementary Fig 2. Validation of GSTO1 knockout A, Western blot probing GSTO1 in nt WT and GSTO1 KO cells after 10, 20, 30 passages. B, Reverse transcriptional real time PCR detection of GSTO1 in nt WT and GSTO1 KO single clones. C, Sanger sequence of GSTO1 KO cell lines Supplementary Fig 3. 3-dimension spheroids of (A) HCT116 nt WT and GSTO1 cells, (B) U-87 MG nt WT and GSTO1 cells Supplementary Fig 4. Neurospheroid formation of U-87 MG nt WT and GSTO1 KO single clones. Supplementary Fig 5. Genes and gene sets up- or down-regulated in U-87 MG, HCT116 and A172 as a result of GSTO1 KO in Bru-seq. A, Genes up- or down- regulated in U-87 MG, HCT116 and A172 as a result of GSTO1 KO. B, Top 10 significant enrichment gene sets modulated by GSTO1 KO in enrichment analysis (GSEA). Supplementary Fig 6. GSEA plots for highlighted significantly changed gene sets. A, GSEA plots for common significantly changed gene sets overlapped in nascent (Bru- seq) transcriptomic profiles between HCT116 and U-87 MG cells in response to GSTO1 KO. B, GSEA plots for UV response gene sets in nascent (Bru-seq) transcriptomic profiles in U-87 MG GSTO1 KO cells. C, GSEA plots for epithelial mesenchymal transition gene sets in nascent (Bru-seq) transcriptomic profiles in U-87 MG GSTO1 KO cells. D, GSEA plots for interferon production and response gene sets in nascent (Bru- seq) transcriptomic profiles in HCT116 GSTO1 KO cells. E, GSEA plots for tight junction, apical junction and Myc gene sets in nascent (Bru-seq) transcriptomic profiles in response to GSTO1 KO. NES = normalized enrichment score; FDR= false discovery rate Supplementary Fig 7. DAVID analysis of significantly changed genes in RNA-seq. 24 gene sets were enriched in both HCT116 and U-87 MG cells. Supplementary Fig 8. Differential activity of cisplatin, carboplatin and oxaliplatin in HCT116 nt WT and GSTO1 KO cell line Supplementary Fig 9. Differential activity of select drugs in HCT116 nt WT and GSTO1 KO cell line. First concentrations used for each compound are illustrated and all the compounds were 2-fold diluted. Compounds showing differential activities are highlighed in red. Supplementary Fig 10. Colony formation of U-87 MG and A172 nt WT and GSTO1 KO cells upon treatment with temozolomide. 500 cell/wells of U-87 MG nt WT, A172 nt WT and GSTO1 KO cells and 2000 cells/well of U-87 MG GSTO1 KO cells were seeded into 96 well plates. Indicated concentration of temozolomide was added the next day. Cells were stained with crystal violet after 7 days of temozolomide treatment. Supplementary Fig 11. GSTO1 KO cells are more sensitive towards radiation. 500 cell/wells of U-87 MG nt WT and GSTO1 KO cells were seeded into 96 well plates. The next day, cells were radiated with indicated dose of radiation. MTT of the cells were determined after 7 days. Supplementary Fig12. RNA transcription of all GSTO1 isoforms is downregulated and protein production is attenuated due to the CRISPR/CAS9 genome editing. A, Isoform expression of GSTO1 in CRISPR treated cell lines in RNA-seq. Bars show mean expression across 3 biological replicates. Error bars represent standard error. B, Abundance ratio of each peptide of GSTO1 probed by protein mass-spectrometry. Triplicate for each sample was used. Supplementary Fig13. Strong agreement of significantly changed gene sets among GSTO1 KO, siGSTO1 KD and GSTO1 inhibitor C1-27 treatment in HCT116 cells. Significantly changed gene sets (GSEA) overlapped among the three treatment in HCT116 cells. Gene sets with FDR<0.1 were considered significant. Supplementary Fig14. Knockdown GSTO1 did not change growth rate of HCT116 cells. HCT116 shScramble and shGSTO1 cells were seeded into 96 well plate at 200 cells/well after treatment with doxycycline for 3 days. Doxycycline was added to the cells at day 2 and day 4. Colony formation and MTT assays were determined at day 7. Supplementary Fig15. HCT116 cells grow slower after GSTO1 knockout or knockdown in medium without glutamine. A. GSTO1 KO cells were seeded into 96 well plate at 200 cells/well in medium with or without glutamine. Cell growth was determined using MTT after 7 days of treatment. B. HCT116 shScramble and shGSTO1 cells were seeded into 96 well plate at 200 cells/well in RPMI without glutamine. Doxycycline was added to the cells at day 2 and day 4. Colony formation and MTT assays were determined at day 7. Supplemental tables Table 1. Top 30 up-regulated genes in U-87 MG GSTO1 KO Bru-seq gene ensemblGene fc log2fc RNASE4 ENSG00000258818.3 251.8141 7.976215 ALG9 ENSG00000086848.14 20.21923 4.337656 TMEM249 ENSG00000261587.2 13.96941 3.804199 AGRN ENSG00000188157.14 9.409367 3.234098 KRT80 ENSG00000167767.13 9.21308 3.203684 SLC9A3R1 ENSG00000109062.11 8.763223 3.131462 SGSM3 ENSG00000100359.20 6.984794 2.804218 LAMA5 ENSG00000130702.15 6.262307 2.646694 CD9 ENSG00000010278.12 6.0346 2.593258 IFITM10 ENSG00000244242.1 5.924491 2.566691 NUPR1 ENSG00000176046.8 5.795572 2.534951 PDK2 ENSG00000005882.11 5.568363 2.477253 TNFAIP2 ENSG00000185215.8 5.491794 2.457277 RRAD ENSG00000166592.11 5.455361 2.447675 TRIB3 ENSG00000101255.10 5.429262 2.440756 HIST1H2BI ENSG00000278588.1 5.301592 2.406426 MKNK2 ENSG00000099875.14 5.290849 2.403499 TXNDC5 ENSG00000239264.8 5.146783 2.363671 DDIT4 ENSG00000168209.4 5.113496 2.35431 HSPA1B ENSG00000204388.6 4.932361 2.302279 NPFF ENSG00000139574.8 4.90164 2.293265 PDGFA ENSG00000197461.13 4.850207 2.278046 MCAM ENSG00000076706.16 4.837727 2.274329 ADAM8 ENSG00000151651.15 4.771018 2.254297 MXD4 ENSG00000123933.16 4.767134 2.253122 LIME1 ENSG00000203896.9 4.595306 2.200161 MIEF2 ENSG00000177427.12 4.525257 2.178 CYBA ENSG00000051523.10 4.470379 2.160397 SAMD11 ENSG00000187634.11 4.443104 2.151568 CHAC1 ENSG00000128965.11 4.429486 2.147139 Table 2. Top 30 down-regulated genes in U-87 MG GSTO1 KO Bru-seq gene ensemblGene fc log2fc IPO4 ENSG00000196497.16 -88.72 -6.471 CHMP4A ENSG00000254505.9 -17.68 -4.144 ADAMTS18 ENSG00000140873.15 -15.268 -3.932 C8orf48 ENSG00000164743.4 -11.636 -3.541 RARB ENSG00000077092.18 -10.189 -3.349 NR0B1 ENSG00000169297.7 -7.8689 -2.976 THBS1 ENSG00000137801.10 -6.842 -2.774 NID2 ENSG00000087303.17 -6.7075 -2.746 VGLL3 ENSG00000206538.8 -6.5068 -2.702 SP140 ENSG00000079263.18 -6.2005 -2.632 SIGLEC15 ENSG00000197046.11 -5.9444 -2.572 LMO7 ENSG00000136153.19 -5.7832 -2.532 EFNB2 ENSG00000125266.6 -5.5506 -2.473 KCTD4 ENSG00000180332.6 -5.4216 -2.439 RNF133 ENSG00000188050.2 -4.9113 -2.296 CD274 ENSG00000120217.13 -4.8502 -2.278 U2AF1L4 ENSG00000161265.14 -4.7602 -2.251 CLN5 ENSG00000102805.14 -4.6937 -2.231 BCL11B ENSG00000127152.17 -4.643 -2.215 FAP ENSG00000078098.13 -4.2768 -2.097 MFSD14A ENSG00000156875.13 -4.2656 -2.093 UQCR10 ENSG00000184076.13 -4.1306 -2.046 GLIPR1 ENSG00000139278.9 -4.0732 -2.026 TMEM156 ENSG00000121895.7 -3.9291 -1.974 GNG2 ENSG00000186469.8 -3.9228 -1.972 HIST1H2BB ENSG00000276410.3 -3.8859 -1.958 NOVA1 ENSG00000139910.19 -3.7983 -1.925 GPC6 ENSG00000183098.10 -3.7794 -1.918 RASD2 ENSG00000100302.6 -3.7052 -1.89 PHOSPHO2 ENSG00000144362.11 -3.6268 -1.859 Table 3. Top 30 up-regulated genes in HCT116 GSTO1 KO Bru-seq gene ensemblGene fc log2fc ZBTB9 ENSG00000213588.5 7.21285 2.8506 EEF1G ENSG00000254772.9 7.21285 2.8506 LIME1 ENSG00000203896.9 6.63398 2.7299 ZNF177 ENSG00000188629.11 5.7496 2.5235 PAF1 ENSG00000006712.14 5.56774 2.4771 DHRS2 ENSG00000100867.14 5.08547 2.3464 IFI6 ENSG00000126709.14 5.04561 2.335 OASL ENSG00000135114.12 4.8795 2.2867 PKIB ENSG00000135549.14 4.7552 2.2495 TNNT1 ENSG00000105048.16 4.5342 2.1808 IK ENSG00000113141.17 4.38286 2.1319 IFIT1 ENSG00000185745.9 4.36136 2.1248 DAXX ENSG00000204209.11 4.27473 2.0958 OS9 ENSG00000135506.15 4.18806 2.0663 AKAP12 ENSG00000131016.16 4.16955 2.0599 MDP1 ENSG00000213920.8 3.98046 1.9929 PTMA ENSG00000187514.16 3.94896 1.9815 TMEM200A ENSG00000164484.11 3.71201 1.8922 CAMK2D ENSG00000145349.16 3.69695 1.8863 SMARCC2 ENSG00000139613.11 3.59108 1.8444 FAM50A ENSG00000071859.14 3.56696 1.8347 OPTN ENSG00000123240.16 3.55945 1.8317 AHNAK2 ENSG00000185567.6 3.51105 1.8119 PALM3 ENSG00000187867.8 3.43693 1.7811 NAV3 ENSG00000067798.14 3.39743 1.7644 RAB13 ENSG00000143545.8 3.37027 1.7529 FIP1L1 ENSG00000145216.15 3.33534 1.7378 HIRIP3 ENSG00000149929.15 3.28724 1.7169 DDX23 ENSG00000174243.9 3.28395 1.7154 CA11 ENSG00000063180.8 3.25335 1.7019 Table 4. Top 30 down-regulated genes in HCT116 GSTO1 KO Bru-seq gene ensemblGene fc log2fc NR5A2 ENSG00000116833.13 -9.1106 -3.188 SYTL2 ENSG00000137501.17 -7.9522 -2.991 F3 ENSG00000117525.13 -5.6306 -2.493 EHF ENSG00000135373.12 -5.0044 -2.323 CCDC89 ENSG00000179071.4 -4.9407 -2.305 RGS2 ENSG00000116741.7 -4.5345 -2.181 ZC2HC1B ENSG00000118491.9 -4.5219 -2.177 CENPS ENSG00000175279.21 -4.4277 -2.147 BDNF ENSG00000176697.18 -4.166 -2.059 BST2 ENSG00000130303.12 -4.1451 -2.051 TMEM249 ENSG00000261587.2 -4.1451 -2.051 SRGAP1 ENSG00000196935.8 -4.1417 -2.05 NECTIN3 ENSG00000177707.10 -3.8507 -1.945 BMP4 ENSG00000125378.15 -3.7877 -1.921 OVCA2 ENSG00000262664.2 -3.3915 -1.762 WLS ENSG00000116729.13 -3.3552 -1.746 PCSK9 ENSG00000169174.10 -3.2892 -1.718 CDRT4 ENSG00000239704.10 -3.2658 -1.707 BBS10 ENSG00000179941.6 -3.204 -1.68 CKLF ENSG00000217555.12 -3.1821 -1.67 HIST1H2AK ENSG00000275221.1 -3.0832 -1.624 ZNF649 ENSG00000198093.10 -3.006 -1.588 ARRDC4 ENSG00000140450.8 -2.9366 -1.554 TMEM86B ENSG00000180089.5 -2.8776 -1.525 TAS2R13 ENSG00000212128.2 -2.8733 -1.523 SDHAF1 ENSG00000205138.3 -2.7347 -1.451 CRIM1 ENSG00000150938.9 -2.7175 -1.442 SYBU ENSG00000147642.16 -2.7007 -1.433 DHFR2 ENSG00000178700.7 -2.6662 -1.415 TLCD1 ENSG00000160606.10 -2.6301 -1.395 Table 5.
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