Fig. 2: Knockout of Bax and Bak compromises clonogenic potential of tetraploid cells.

Mouse embryonic fibroblasts (MEFs) (A–D), human colon carcinoma HCT116 cells (E), or immortalized MEFs derived from C57Bl/6 mice (MEF C57Bl/6) (F) were seeded on day 0, cultured from day 1 to day 3 (48 h) in drug-free medium or treated with 100 nM Nocodazole (Noco) (A–D and F) or 1.2 μM cytochalasin D (CytD) (A, E), then washed and kept in a drug-free medium for 4 more days, and finally sorted on day 7 (100 cells per well). Diploid (2n) cells were derived from cells grown in a drug-free medium (A–C, E, F) or from cells treated with CytD (E), whereas polyploid (>4n) cells were derived from cells treated with Noco (A–D and F) or CytD (E). The cell cycle and the DNA content gates were established by cell staining with 10 μM Hoechst 33342 for 1 h (B). Clonogenicity of MEF (C), HCT116 (E), or MEF C57Bl/6 (F) cells deficient for one or two pro-apoptotic proteins was quantified at day 21 as percentage of wells in which there was cell proliferation. Alternatively, >4n wild type (WT) and Bax^−/−Bak^−/− (DKO) MEFs sorted on day 7 were subjected for cell proliferation assay at different time points between days 7 and 21 (D). Bars with blue contours represent WT cells and bars with red contours represent Bax/Bak DKO cells. White columns represent clonogenicity of 2n cells and black columns represent clonogenicity of >4n cells. Error bars indicate SEM. Data were compared by R software using standard linear model inference, “lm” function. >4n Samples were compared to 2n samples for each cell line (##p < 0.01, ###p < 0.001 vs. 2n). Clonogenicity of >4n cells was compared between all MEFs deficient for different pro-apoptotic proteins vs. DKO cells (p-values in red, C); or between DKO (“D”) and WT (“W”) cells (E, F) (*p < 0.05, **p < 0.01). Cell proliferation of >4n DKO (red) vs. >4n WT (blue) MEFs (D) was compared (**p < 0.01).