Supernumerary centrosomes and chromosomal instability promoted by pRb acute loss were not affected by p53 status. A) RT-PCR (top panel) and Western blot (bottom panel) showing RB mRNA and protein levels, respectively, at 72 hours post-transfection in both wild type and p53 knockout HCT116 cells. RT-PCR revealed the presence of RB mRNA in both untransfected cell lines, (lane 2: HCT116-wt, lane 4: HCT116p53KO) but not in RB-depleted cells (lane 3: HCT116-wt, lane 5: HCT116p53KO). Amplification of GAPDH (330 bp) was used as control of the quality of the cDNA. The 100 bp DNA-ladder was loaded in lane 1 as a size marker. Western blot confirmed selective depletion of pRb in siRNA-transfected cells (lane 2: HCT116-wt, lane 4: HCT116p53KO) in comparison with untransfected cells (lane 1: HCT116-wt, lane 3: HCT116p53KO). β-tubulin was used as a loading control. B) Graphs (top panels) showing percentage of cells with 1, 2 or more than 2 (>2) centrosomes in untransfected (untr.), RB-depleted (siRB) and released (release) HCT116-wt and HCT116p53KO. Presence of supernumerary centrosomes (bottom panels, white arrow) detected by γ-tubulin immunostaining (green) in both HCT116-wt and HCT116p53KO cells after pRb acute loss (a, b, respectively). A magnification for each cell type is reported on the right. Nuclei were stained with DAPI (blue). C) Representative pictures of diploid (2N), hypodiploid (<2N) and hyperdiploid (>2N) metaphases in both HCT116-wt and HCT116p53KO pRb-depleted cells. D) Graph summarizing the percentages of diploid (2N), hypodiploid (<2N) and hyperdiploid (>2N) metaphases observed in the indicated cell types transfected with siRNA targeting RB (siRb), untransfected (untr.) and released 10 days (release).