Fig. 4: Loss of SIN3B does not phenocopy LIN37 deficiency in T98G cells.

a A CRISPR/Cas9-nickase approach to introduce mutations in exon 4 of SIN3B and exon 6 of LIN37 was applied to generate cell lines negative for SIN3B, LIN37, or both proteins. SIN3B knockout clones were confirmed with antibodies targeting amino acids 172-228 (SIN3B-H4) or amino acids 668-758 (SIN3B polyclonal). LIN37 knockout was confirmed with a polyclonal antibody raised against full-length LIN37. b mRNA expression of G2/M (BUB1, CCNB2, BIRC5) and G1/S (MCM5, ORC1) cell-cycle genes was analyzed by RT-qPCR in wild-type and knockout lines arrested by serum-starvation for 48 and 96 hours. Two independent SIN3B^-/-, LIN37^-/-, and SIN3B^-/-;LIN37^-/- clones were compared with two wild-type (WT) clones. Averages of two biological replicates measured with two technical replicates each are given. c Protein expression of one of the wild-type and knockout clones measured in (b) was analyzed by Western blotting. Samples were derived from the same experiment and blots were processed in parallel. Similarly, d mRNA expression and e protein levels of cell-cycle genes were analyzed in two wild-type or knockout lines treated with 10 µM Palbociclib for 24 or 48 hours. f Indicated wild-type and knockout lines (two clones each) were serum-starved for 96 hours with or without 10 µM Palbociclib for the final 48 hours. mRNA was measured (two biological replicates with two technical replicates each) and compared with untreated wild-type mRNA levels. g Samples shown in (f) were analyzed for protein expression by Western blotting. h Cell-cycle distribution of T98G wild-type (WT) and knockout lines was analyzed by DNA staining with propidium iodide and flow cytometry. Two independent clones for each line were measured with three biological replicates. i HDACI/II activity of samples immunoprecipitated from T98G wild-type and knockout cells serum-starved for 96 hours with the indicated antibodies. Each data point contains four technical replicates of a representative experiment. Two biological replicates produced similar results. j Protein expression and immunoprecipitation efficiency of the samples analyzed in (i) were evaluated by Western blotting. Data in Figs. (b), (d), (f), (h), and (i) are given as mean values ± SEM, and significances were calculated with the two-tailed Student’s T-Test (ns – not significant, * p ≤.05, ** p ≤ .01, *** p ≤ .001). At least two biological replicates were performed for each Western blot experiment, and the results were similar. Source data are provided as a Source Data file.