Additional file 7 of Dissecting super-enhancer driven transcriptional dependencies reveals novel therapeutic strategies and targets for group 3 subtype medulloblastoma
Creators: name: Li, Meng
name: Han, Yujie
name: Wang, Chaochen
name: Kang, Wenfeng
name: Jiang, Wenyan
name: Zhang, Lei
name: Tang, Yujie
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 7: Fig. S1. a-b Violin plots showing GSVA score of cSE (a) or tSE (b) signature genes in four MB subgroups or NC of the indicated MB datasets. (c-d) Kaplan-Meier survival analysis of the GSVA scores of cSE (c) or tSE (d) genes in Cavalli dataset of MB. The patient cohort was stratified as high versus low groups by median GSVA score. Statistical significance was determined by one-way ANOVA (a-b), two-sided log-rank test (c-d). Fig. S2. a-c RT-qPCR analysis of vSE genes in MB002 or D425 cells when MYC (a), OTX2 (b) and CRX (c) were knocked down by shRNA individually. shSCR served as control. RT-qPCR assays were performed in triplicate and the data are presented as mean ± SD. Statistical significance was determined by two-tailed unpaired t test (a-c). Fig. S3. a Gene tracks of H3K27Ac ChIP-seq signal across G3-MB cells and tissues at SE regions near PSMB5. SE region is lined out over the gene tracks. b Gene tracks of H3K27Ac ChIP-seq signal across five G3-MB lines and non-G3-MB cells UW228 at SE regions near PSMB5. SE regions are lined out over the gene tracks. Positions of tested ChIP-qPCR amplicons, pooled-CRISPRi sgRNA targeting regions and 3C-PCR primers are shown below the tracks. c Box plots showing the FPKM values of PSMB5 from RNA-Seq replicates of D283, D425 and MB002 lines versus UW228, a control non-G3-MB line. d ChIP-qPCR analysis of H3K27Ac enrichment near PSMB5 in MB002, D283 and UW228, respectively. e 3C-PCR analysis of chromatin interaction between the H3K27Ac peak regions and promoter of PSMB5 in MB002, D283 and UW228. The workflow of 3C-PCR was shown in top panels, the agarose gel electrophoresis of 3C-PCR products was shown in bottom panels. The positions of 3C-PCR primers are shown in (b). f Negative control SE region of PSMB5 (NCP) without MboI site of 3C-PCR analysis related to Fig. S3 (e) was tested in MB002, D283 and UW228 by RT-qPCR. g-h Pooled-CRISPRi sgRNAs are designed to target the SE regions of PSMB5 in stable dCas9-KRAB expressing MB002 and D283, the PSMB5 expression level (g) is measured by RT-qPCR, and cell viabilities (h) are measured at Day 6. i RT-qPCR analysis of MYC and PSMB5 in D283 cells treated with 1 μM JQ1 or 0.1 μM THZ1 for 6 h. RT-qPCR analysis of PSMB5 in D283 when MYC (j), OTX2 (k) and CRX (l) were knocked down by shRNA individually (left). The cell viabilities were also measured (right). All RT-qPCR and cell viability assays were performed in triplicate and the data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA (d) and two-tailed t test (c) and (g-i). Fig. S4. a UMAP plot showing MB subtypes (top left) or expression levels of PSMB5 in MB (top right) and GP3-MB (middle left), MYC (middle right), OTX2 (bottom left) and CRX (bottom right) in GP3-MB. Fig. S5. a Cell viability (top) and CI (bottom) of MB002 and D425 cells treated with THZ1 and Marizomib individually or in combination at the indicated concentrations for 72 h. b Cell viability (top) and CI (bottom) of D425 cells treated with JQ1 and Marizomib individually or in combination at the indicated concentrations for 72 h (top). c Cell viability of MB002 and D425 cells treated with Marizomib (3.25 nM for MB002, 125 nM for D425) and JQ1 (500 nM) individually or in combination were measured at Day 0/2/4 post treatment and normalized to Day 0-value. d The body weight of xenograft nude mice described in Fig. 4 (f-h) was recorded. Cell viability assays were performed in triplicate and the data are presented as mean ± SD. Line plot showing body weight of xenograft nude mice was presented as mean ± SEM in (d). Fig. S6. a UMAP plot showing MB subtypes (top left) or expression levels of ARL4D in MB (top right), GP3-MB (middle left) and GP4-MB (middle right), OTX2 (bottom two rows, left) and CRX (bottom two rows, right) in GP3- or GP4-MB. Fig. S7. a-b MB002 and D425 cells were infected with Cas13d empty vector (EV) or two separate clones of Cas13d-sgARL4D lentiviruses individually. After puromycin selection, the infected cells were then subjected to RT-qPCR analysis of ARL4D (a) as well as cell viability measurement at Day 0/2/4 (b). Uninfected tumor cells (Mock) were analyzed in parallel. RT-qPCR and cell viability results were normalized to EV or Day 0 sample, respectively. c UW228 cells were infected with Cas13d empty vector (EV) or two separate clones of Cas13d-sgARL4D lentivirus individually at the same MOI as MB002 and D425 in (a-b). Cell viability were measured at Day 0/2/4 and normalized to Day 0 sample. d-e FACS analyses of cell proliferation (d) and apoptosis (e) of MB002 and D425 cells upon ARL4D knockdown with Cas13d-sgARL4D. All RT-qPCR and cell viability assays were performed in triplicate and the data are presented as mean ± SD. Fig. S8. a Percentage ranking analysis of ARL4D as SE- or TE-target gene in five G3-MB cells and non-G3-MB cells UW228 was shown in the table. b Negative control SE region of ARL4D (NCA) without HindIII site of 3C-PCR analysis related to Fig. 6 (d) was tested in MB002 and UW228 by RT-qPCR. c-e 3C-PCR analysis of chromatin interaction between the H3K27Ac peak regions and promoter of the ARL4D in MB002. The positions of 3C-PCR primers are shown in (c). The same NCA region as above (neither containing the MboI site) was tested by RT-qPCR (d). The workflow of 3C-PCR was shown over the electrophoretogram of 3C-PCR products (e). f ChIP-qPCR analysis of OTX2 (top) and H3K27Ac (bottom) enrichment near ARL4D in MB002 upon OTX2 knockdown. g Phase changes of cell cycle in MB002 and D425 following ARL4D knockdown with two separate Cas13d-sgARL4D clones were determined by FACS as described in Fig. 7 (i). h The frequencies of tumorsphere-forming cells of MB002 and D425 upon ARL4D knockdown with two separate shRNA clones were determined by limiting dilution assay as described in Fig.7 (j) and analyzed by L-CalcTM software. ChIP-qPCR assay was performed in triplicate and the data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA (f).