Fig. 2

Effects of REST knockdown on gene expression in IDH-WT and IDH-MUT U87 cells. A Sorted values of log2 fold change (log2 FC) for DEGs in the IDH-MUT vs IDH-WT comparison in U87 cells are presented as red dots. Values for the same genes inferred from the IDH-MUT vs IDH-WT comparison in TCGA G2/G3 gliomas are overlaid as gray dots. Percentage of log2 FC direction concordance between IDH-MUT vs IDH-WT in U87 cells and glioma tumors was calculated. A number of DEGs is indicated. Black vertical line separates genes with higher expression in IDH-MUT U87 versus IDH-WT U87 from the genes with higher expression in IDH-WT U87 versus IDH-MUT U87. B Relative expression of REST in IDH-WT and IDH-MUT U87 cells at 72 h of REST silencing with siRNA. mRNA levels in transfected cells were determined with quantitative PCR and normalized to GAPDH expression in the same sample. Data are represented as mean ± SEM, n = 4 independent experiments, *p < 0.05, two-tailed Mann–Whitney test (WT: p = 0.0286; MUT: p = 0.0286). C Levels of REST protein in IDH-WT and IDH-MUT U87 cells at 72 h after transfection with control or REST specific siRNAs determined with Western blotting. Immunoblots were analyzed by densitometry. Data are represented as mean ± SEM, n = 4. **p < 0.01 (WT: p = 0.0018, MUT: p = 0.0052; two-tailed ratio-paired t-test). No difference was observed in the level of REST protein between IDH-MUT and IDH-WT controls (siCTRL) (p = 0.226). D Volcano plots of the genes differentially expressed between siCTRL and siREST-transfected IDH-WT (upper plot) or IDH-MUT (bottom plot) U87 glioma cells. The axes show log2 fold change (x-axis) and -log10 from adjusted q-value (y-axis). E Gene Ontology (GO) Biological Processes (BP) analysis was performed on DEGs common in IDH-WT and IDH-MUT U87 cells. The results are presented as bar plots for pathways upregulated (upper panel) and downregulated (bottom panel) in REST depleted cells