Fig. 5: Sin3b knockout does not phenocopy loss of Lin37 in mouse C2C12 cells.

a A CRISPR/Cas9-nickase approach was applied to generate cell lines negative for Sin3b. Sin3b knockout clones were confirmed with antibodies targeting an epitope within amino acids 172-228 (SIN3B-H4). Lin37^-/- C2C12 cells were described before (Mages et al. 2017). b mRNA expression of cell-cycle genes was analyzed by RT-qPCR in wild-type and knockout lines arrested by serum starvation over 48 and 72 hours. Two wild-type, two Lin37^-/-, and two SIN3B^-/- clones were measured with two biological and two technical replicates each. c Cell-cycle distribution of C2C12 wild-type (WT) and Sin3b knockout lines was analyzed by DNA staining with propidium iodide and flow cytometry. Two independent clones for each line were measured with two biological replicates. d mRNA expression of cell-cycle genes was analyzed by RT-qPCR in the same wild-type and knockout lines shown in (b) treated with 5 µM Idasanutlin for 24 and 48 hours. e Same experimental setup as in (c), but cells were arrested with Idasanutlin instead of serum-starvation. f The protein expression of one clone analyzed in (d) was studied by Western blot. Data in (b), (c), (d), and (e) are presented as mean values ± SEM. Significances were calculated with the two-tailed. Student’s T-Test (ns – not significant, * p ≤.05, ** p ≤ .01, *** p ≤ .001). Western blot experiments were performed with at least two biological replicates with similar results. Source data are provided as a Source Data file.