Fig. 3

Loss of DIP2C affects RKO cell growth and cell cycle progression and induces senescence markers. a Colony formation assay for DIP2C knockout cells. Left, representative images of wells. Right, quantification of the mean plating efficiency of two independent experiments. All samples were normalized to the parental RKO used in each experiment. Error bars, SD. b Growth curve for DIP2C knockout cells. Cells seeded at equal density were harvested and counted once per day. Mean fold increase compared to the number of seeded cells and SD of technical duplicates is shown. The experiment was repeated at least twice for each clone. c Growth curve for RKO cells overexpressing wild type DIP2C showing mean cell confluency from daily imaging in an IncuCyte instrument. Error bars, SD of four replicate wells. d Cell cycle distribution determined by FACS. Error bars, SD of two independent experiments. e Western blot for p53 and p21 expression. Cell lines and targets as indicated. f Quantification of p53 and p21 immunoblots showing the mean of two independent experiments normalized to the β-actin loading control. Error bars, SD. g-h Quantification of expression from the CDKN2A locus by RT-qPCR with primers designed to target either multiple CDKN2A isoforms, or specifically p14 ^ARF or p16 ^INK4a. Error bars - min and max relative quantity (RQ). g Mean of two biological DIP2C ^−/− #1-1 replicates and one DIP2C ^−/− #1-2 sample normalized to parental RKO. h Expression of all CDKN2A isoforms in DIP2C overexpressing (OE) cells. i Staining for β-galactosidase activity at pH 6. Quantification shows mean fraction of positive cells in six images per sample, at least 2000 cells were counted. Error bars -SD. Representative micrographs, blue – positive stain. i Morphology of live cells under normal growth conditions. Scale bar 100 μm. Results for DIP2C ^−/− #1-1 and DIP2C ^+/− #1 are displayed in figure unless otherwise indicated (*P < 0.05; **P < 0.01; ***P < 0.001; two-tailed Student’s t-test)