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SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure S1. RBPJ Xie et al. SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure S1. RBPJ correlates with BTIC marker expression. A-D. The TCGA GBM dataset was downloaded and correlations analyzed by R. RBPJ mRNA levels were highly correlated with (A) Olig2, (B) Sox2, (C) CD133, and (D) Sox4 levels. E. RBPJ is preferentially expressed in proneural glioblastomas. The glioblastoma TCGA dataset was interrogated for RBPJ mRNA expression segregated by transcriptional profile. The proneural tumors were further divided into G-CIMP (glioma CpG-island methylator phenotype) or non-G-CIMP. **, p < 0.01. ****, p < 0.0001. *****, p < 0.00001. Supplemental Figure S2. Targeting RBPJ induces BTIC apoptosis. A. 3691 BTICs were transduced with shCONT, shRBPJ-1, or shRBPJ-2. Lysates were prepared and immunoblotted with the indicated antibodies. shRNA-mediated knockdown of RBPJ was associated with increased cleaved (activated) PARP. B. 3691 BTICs were transduced with shCONT, shRBPJ-1, or shRBPJ-2. Apoptosis measured by Annexin V staining. Data are presented as mean ± SEM (two- way ANOVA; **, p < 0.01; n = 3). Supplemental Figure S3. Targeting RBPJ does not affect non-BTIC proliferation. Non-BTICs (Top, 3691; Bottom, 4121) were transduced with shCONT, shRBPJ-1, or shRBPJ-2. Cell proliferation was measured by CellTiter-Glo. 42 Xie et al. Supplemental Figure S4. RBPJ induces transcriptional profiles in BTICs distinct from Notch activation. A. In parallel experiments, 3691 BTICs were either treated with DAPT (at either 5 μM or 10 μM) vs. vehicle control (DMSO) or transduced with shRBPJ vs. shCONT. RNA-Seq was performed and the results displayed as a heat map with normalization to the relevant control. Selected genes are displayed based on biologic processes. B. 3691 BTICs were transduced with shCONT, shRBPJ-1, or shRBPJ-2. Gene expression was analyzed by real-time qPCR (two-way ANOVA; **, p < 0.01; n = 3). C. 3691 BTICs were treated with vehicle control (DMSO) or DAPT (5 or 10 μM). Gene expression was analyzed by real-time qPCR (two-way ANOVA; **, p < 0.01; n = 3). Supplemental Figure S5. Transcriptomic profiles in BTICs subjected to RBPJ knockdown, DAPT treatment alone, or combined conditions. A. Ranked list of genes based on their correlation with PC3 for 4121 BTICs based on RNA sequencing after transduction with either shCONT or shRBPJ and treatment with either vehicle control (DMSO) or DAPT treatment. All genes with p < 0.05 were first identified and further narrowed to genes with positive or negative correlations with p < 0.01. B. Genetic programs associated with genes correlated with RBPJ knocked-down in 4121 BTICs. C. Genetic programs associated with genes correlated with RBPJ knocked-down 4121 BTICs treated with DAPT (5 μM). Supplemental Figure S6. RBPJ binds to the FOXM1 and OLIG2 promoters independently from Notch activation. A. Cross-linked chromatin was prepared from 3691 BTICs or Non-BTICs then 43 Xie et al. immunoprecipitated with an anti-RBPJ antibody followed by real-time PCR using primers specific to the indicated regions of FOXM1 promoter (Student’s t-test; n = 3). B. RNA expression (by FPKM) in 3691 BTICs of stemness genes bound (red) or not bound (blue) to RBPJ. Median, 75 % and 25 % quartiles demarcated for each group. Median for bound genes was FPKM of 64, while it was 5 for non-bound genes. Mean for bound genes was FPKM of 50, while it was 10 for non-bound genes. C. Cross-linked chromatin from 3691 or 4121 BTICs was prepared then immunoprecipitated with an anti-RBPJ antibody followed by real-time PCR using primers specific to the indicated regions of OLIG2 promoter (Student’s t-test; n = 3). D. Cross-linked chromatin was prepared from 3691 BTICs treated with DAPT (5 μM) or vehicle control (DMSO), then immunoprecipitated with an anti-RBPJ antibody, followed by real-time PCR using primers specific to the indicated regions of FOXM1, OLIG2 promoters (n = 3). Supplemental Figure S7. RBPJ binds to CDK9 and LSD1. A. Lysates of 3691 cells transfected with HA-RBPJ were immunoprecipitated with anti-HA antibody followed by mass spectrum analysis. The spectral identification of peptide HENVVNLIEICR (CDK9: 75-86AA) is shown. B. Lysates of 3691 non-BTICs transfected with HA-RBPJ were immunoprecipitated with anti-HA antibody followed by immunoblotting for LSD1. Supplemental Figure S8. c-Myc regulates RBPJ expression. The WashU EpiGenome Brower was interrogated at the promoter region of RBPJ for c-Myc genomic binding in human 44 Xie et al. embryonic stem cells in association with the active transcriptional mark of histone 3 lysine 27 acetylation (H3K27ac). MYC binding co-localized with H3K27ac at the RBPJ locus. Supplemental Figure S9. Ingenuity Pathway Analysis (IPA) of RBPJ in normal/neural stem cells vs. BTIC. RBPJ-interacting proteins were inquired using IPA tools such as Pathway Explorer (Qiagen). Proteins having been show to directly interact with RBPJ or its promoter were queried. Furthermore, pathway discovery tools were used to identify connectivity between normal stem cell and cancer stem cell pathways. Blue: pathways involved in neural stem cells. Red: pathways involved in brain tumors and BTICs. Dotted line: causal relationship. Solid line: direct interaction between factors or with promoters. Supplemental Figure S10. Proposed model of RBPJ function in tumor initiating cells. 45 Figure S1 A B 10 10 8 8 6 6 Olig2 Expression Olig2 SOX2 Expression SOX2 4 4 81012 81012 RBPJ Expression RBPJ Expression R=0.387 R=0.301 P<0.0001 P<0.0001 C D 10 10 8 8 6 6 SOX4 Expression SOX4 CD133 Expression CD133 4 4 7 8 9 10 11 12 8 10 12 RBPJ Expression RBPJ Expression R=0.193 R=0.550 P<0.0001 P<0.0001 E **** **** 12 **** ** 11 10 9 8 mRNA expression (log2) expression mRNA 7 Neural G-CIMP Classical Mesenchymal Proneural Non-G-CIMP Figure S2 A 3691 BTICs RBPJ shRNA CONT #1 #2 RBPJ PARP Actin B 3691 BTICs 60 ** ** 40 20 Annexin V postive cell(%) V postive Annexin 0 2# CONT Sh-RBPJ1# Sh-RBPJ Figure S3 3691 Non-BTICs 4 shCONT shRBPJ#1 3 shRBPJ#2 2 1 Relative cell number Relative cell 0 0 2 4 6 8 Time (days) 4121 Non-BTICs 5 shCONT 4 shRBPJ#1 shRBPJ#2 3 2 1 Relative cell number cell Relative 0 0 2 4 6 8 Time (days) Figure S4 A 0 1.0 2.0 DMSO shRBPJ#1 shCONT shRBPJ#2 DAPT 5 uM DAPT DAPT 10 uM DAPT RBPJ HES1 HES5 HEY1 Canonical Notch downstream targets HEY2 OLIG1 OLIG2 ASCL1 TFs controlling stemness in BTICs FOXM1 MYC BUB1 BUB1B CDC20 CDK1 CCNA2 CCNB2 CCND2 Cell cycle regulators PLK1 PLK4 KRAS NEK2 B Notch target genes Stemness genes Cell Cycle gens shCONT 3 shRBPJ#1 ** shRBPJ#2 2 ** 1 **** ** ** * ** Relative mRNA levelRelative mRNA * ** ** ** 0 MYC BUB1 HES1 HES5 HEY1 HEY2 CDK1 KRAS OLIG1 OLIG2 ASCL1 CDC20 CCNA2 FOXM1 C Notch target genes Stemness genes Cell Cycle gens DMSO 2.0 DAPT 5 uM DAPT 10 uM 1.5 1.0 ** ** 0.5 ** Relative mRNA levelRelative mRNA ** 0.0 MYC HES1 HES5 HEY1 HEY2 BUB1 CDK1 KRAS OLIG1 OLIG2 ASCL1 CDC20 CCNA2 FOXM1 Figure S5 A Correlation (r) BC Intrinsic Vesicle Trafficking Translation Ribosome Apoptosis Semaphorin Biogenesis Pathway Signaling FDR FDR 0.001 0.05 0.001 0.05 Figure S6 A B IgG RBPJ ChIP on FOXM1 promoter RBPJ 100 1.0 *** 80 0.8 60 0.6 40 0.4 ns 1% Input FPKM RNA-Seq FPKM 20 0.2 0 0.0 Stemness Genes BTICs Non-BTICs RBPJ ChIP on OLIG2 promoter by RBPJ Bound Not Bound by RBPJ C D RBPJ ChIP on FOXM1/OLIG2 promoter IgG 1.5 ** RBPJ 1.5 DMSO ** DAPT 1.0 1.0 1% Input 0.5 % Input 0.5 0.0 0.0 3691 4121 FOXM1 OLIG2 Figure S7 A b +2 8 y 460.4 4 b b b b b b b 100 577.32 4 5 6 7 8 9 11 HEN V V N L I E I C R( CDK9 75-86AA) 80 y9 y8 y7 y6 y5 y4 y3 y2 y1 +2 b7 403.8 60 b4 40 y5 690.4 b7 y y +2 8 3 b9 806.5 y9 Relative Abundance Relative y +2 4 +2 524.9 b 20 y5 b5 6 345.8 y7 y +2 b 1 y2 b11 917.5 9 y6 175.0 b8 1048.6 0 200 400 600 800 1000 1200 1400 B 3691 Non-BTICs HA-RBPJ -+ LSD1 IP: HA HA-RBPJ HA-RBPJ Input LSD1 GAPDH Figure S8 WashU EpiGenome Database 26313000 26323000 26333000 26343000 Myc H1 human ES cells ES human H1 H3K27ac p15.2 Chr4 RBPJ Figure S9 Figure S10 Full unedited gel for Figure 1C Full unedited gel Figure 2A 3691 BTICs 4121 BTICs 387 BTICs 3691 BTICs 4121 BTICs 130 130 130 130 130 100 Cleaved Notch1 100 100 100 100 Cleaved Notch1 70 70 70 70 70 55 55 55 40 40 Actin 40 130 130 35 100 100 70 70 RBPJ 55 55 70 55 55 Full unedited gel Figure 2D 40 Actin 100 40 35 70 35 RBPJ 55 40 Full unedited gel Figure 2C 387 3691 4121 70 55 100 GFAP 100 100 40 70 70 70 RBPJ 55 55 55 55 40 55 55 55 Olig2 35 40 40 40 SOX2 35 35 35 55 25 25 25 Tubulin 40 55 55 55 35 40 40 40 Olig2 35 35 35 Full unedited gel Figure 3A, B 100 100 100 3691 BTICs 4121 BTICs 70 70 70 55 55 55 130 GFAP 100 40 40 40 100 70 70 55 RBPJ 55 55 55 55 Actin 40 40 40 40 40 35 35 35 35 GAPDH 35 25 25 15 15 Full unedited gel Figure 6C IP Input Full unedited gel Figure 7A 3691 BTICs 4121 BTICs 55 100 55 55 40 CDK9 70 HA-RBPJ CDK9 40 40 35 55 35 35 55 100 40 CDK9 70 70 70 HA-RBPJ 35 55 55 55 55 Tubulin 40 40 40 35 GAPDH 35 25 Full unedited gel Figure 8B 3691 BTICs 4121 BTICs Full unedited gel Figure 8C 3691 Non-BTICs 4121 Non-BTICs 100 100 100 70 100 70 RBPJ 70 70 RBPJ 55 55 55 55 100 100 70 70 130 Myc 130 55 55 100 100 70 70 Flag- Myc 55 40 40 55 GAPDH 35 35 40 40 GAPDH 25 25 35 35 25 25 Full unedited gel Figure 8D Full unedited gel Figure S7B Full unedited gel Figure S2A 3691 BTICs 4121 BTICs IP Input 100 100 100 180 180 70 70 70 RBPJ 130 130 RBPJ LSD1 55 55 55 100 100 100 100 180 100 100 130 70 70 70 70 HA-RBPJ 100 PARP 55 55 55 Myc 55 70 40 40 40 55 GAPDH 35 35 35 GAPDH 40 Actin 25 25 25 35.
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