Fig. 6: Combined depletion of SIN3A and SIN3B derepresses a subset of cell-cycle genes independently of DREAM or RB.
From: HDAC activity is dispensable for repression of cell-cycle genes by DREAM and E2F:RB complexes
Transcriptome analyses were performed with HCT116 wild-type (WT) and SIN3B-/- HCT116 cells transfected either with a non-targeting siRNA or with SIN3A siRNAs for 48 hours and treated with Idasanutlin for the final 24 hours. a Volcano plots show up and downregulated genes in comparison to wild-type cells. Numbers of significantly regulated genes (p < 0.05, fold change ≥1.5) are shown in red. Genes identified as LIN37/DREAM targets before14 are highlighted in blue. The p-values indicate the probability that the respective overlap between LIN37 and SIN3-regulated genes could be observed by chance (hypergeometric test). b The number and overlap of LIN37 target genes identified in Uxa et al.14 and genes upregulated (p < 0.05; FC ≥ 1.5) in Idasanutlin-treated HCT116 cells depleted of SIN3A, SIN3B, or both proteins. c GO analyzes (biological processes) of significantly upregulated (p < 0.05; FC ≥ 1.5) genes. The top ten hits based on their false discovery rate (FDR, as calculated by ShinyGo95) are shown. Cell-cycle distributions of d HCT116 wild-type and e SIN3B knockout lines were analyzed 48 hours after transfection with a control (CTRL) siRNA or three SIN3A siRNAs and Idasanutlin treatment for the final 24 hours. DNA was stained with propidium iodide and analyzed by flow cytometry. Averages of three biological replicates ± SEM are shown. f Wild-type, SIN3B-/-, SIN3B-/-;LIN37-/-, and SIN3B-/-;RB-/- knockout lines were transfected with non-targeting or SIN3A siRNAs for 48 hours, and Idasanutlin was applied for the final 24 hours. Protein levels were analyzed by Western blotting. A representative blot of cells transfected with either a non-silencing control RNA (CTRL) or SIN3A siRNA 1 is shown. A biological replicate with SIN3A siRNA 4 produced similar results. g mRNA levels of genes identified as significantly upregulated in SIN3A/B-depleted cells in the transcriptome analysis were evaluated by RT-qPCR. Cells were treated as described in (f) but additionally transfected with SIN3A siRNA 4. Averages (mean values ± SEM) of three biological and two technical replicates are given. Significances were calculated with the two-tailed Student’s T-Test (ns – not significant, * p ≤.05, ** p ≤ .01, *** p ≤ .001). Source data are provided as a Source Data file.
