Figure 2

pRb depleted cells overrode mitosis even when chromosome mis-segregation occurred. A) Immunodetection of β-tubulin (green) in pRb depleted cells, wild-type (top) and p53-KO (bottom), showing mitotic defects (white arrows). Nuclei were counterstained with DAPI (blue). B) Centromere immunostaining with a CREST antibody detected the presence of centromeres (green) in micronuclei (white arrow) after pRb acute loss in both HCT116 wild-type (top) and p53-KO (bottom) cells. Nuclei were counterstained with DAPI (blue). C) Graph showing the percentage of HCT116 cells with 1, 2 or more than 2 centrosomes at 72 hours post-transfection of siRNAs targeting p53, alone or in combination with siRNAs targeting RB. D) Graph summarizing the percentages of diploid (2N), hypodiploid (<2N) and hyperdiploid (>2N) metaphases at 72 hours post-transfection of siRNAs targeting p53, alone or in combination with siRNAs targeting RB. p53 depleted (sip53) and pRb/p53 co-depleted (siRB/p53) cells showed a similar increase in the percentage of hypodiploid metaphases in comparison with untransfected cells. E) Immunofluorescence microscopy detecting γ-H2AX foci (green, red arrows) in wild-type (HCT) and p53 knockout (HCTp53KO) pRb-depleted cells after 24 hours of doxorubicin treatment (siRB+Doxo). In contrast, only background signals were detected in untreated RB-depleted (siRB) or untransfected (untr) cells. Nuclei were counterstained with DAPI (blue). F) Western blot analysis did not show increased γ-H2AX levels in HCT and HCTp53KO cells after pRb acute loss (+ siRB) in comparison to untransfected cells (-siRB, top). On the contrary, after doxorubicin (Doxo) treatment γ-H2AX protein levels increased in both cell types (bottom). β-tubulin was used as a loading control.