Fig. 7
ARL4D is required for maintaining cell cycle progression and inhibiting neural differentiation of G3-MB cells. a Volcano plots showing significantly altered genes (mean FPKM of shSCR or shARL4D ≥ 1, log2_FC < -1 or > 0.6, FDR < 0.05) in MB002 cells upon ARL4D knockdown by two separate shRNA clones. Selected cell cycle and neural development related genes for further validation are shown. b-c Venn diagram analysis of significantly downregulated (b, mean FPKM of shSCR ≥ 1, log2_FC < -1, FDR < 0.05) or upregulated genes (c, mean FPKM of shARL4D ≥ 1, log2_FC > 0.6, FDR < 0.05) in MB002 cells upon ARL4D knockdown by two separate shRNA clones. d-e GO (BP, biological processes) and Pathway (KEGG and REACTOME) analyses of the shared downregulated (d) or upregulated genes (e) identified in (b) and (c), respectively. f Heatmap of gene expression levels of the selected cell cycle and neural development related genes that are significantly downregulated or upregulated upon ARL4D knockdown in MB002 cells by two separate shRNA clones. g-h RT-qPCR validation of the selected significantly differentially expressed cell cycle (g) or neural development (h) related genes tested in (f) upon ARL4D knockdown by two separate shRNA clones in MB002 and D425 cells, respectively. i FACS analysis of cell cycle of MB002 and D425 cells with ARL4D being knocked down following infection of two separate clones of Cas13d-sgARL4D lentivirus. Tumor cells stably expressing Cas13d empty vector (EV) and uninfected tumor cells (Mock) were analyzed in parallel as control. j Limiting dilution analysis of the frequency of tumorsphere forming cells of MB002 and D425 cells following ARL4D knockdown by two separate shRNA clones. All RT-qPCR assays were performed in triplicate and the data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA (g-h)