Fig. 7. p21-PCNA interaction is detrimental for cell survival after UV irradiation. (A) U2OS cells transfected with empty vector (EV) or 6Mycp21 (CDK–) were UV irradiated (10J/m²). After fixation, immunostaining with phosphorylated H2AX (H2AX)- and p21-specific antibodies was performed. DAPI staining was used to visualize the nucleus. A complete panel showing all p21 mutants is shown in supplementary material Fig. S5. (B) U2OS cells transfected with the indicated plasmids were irradiated with 10J/m². The number of cells with H2AX accumulation was quantified for each time point. The data shown represent the percentage of p21-positive cells with detectable accumulation of H2AX. In all cases, 100 transfected nuclei were counted. The last column in each group corresponds to the percentage of total cells with detectable accumulation of H2AX. Values are the average and error bars are the standard deviation between equivalent samples in two independent experiments. (C) U2OS cells transfected with EV or 6Mycp21 (CDK–) and GFP-pol were UV irradiated utilizing polycarbonate filters and H2AX staining used to detect the irradiated areas. (D) U2OS cells transfected with GFP-PCNA and the indicated p21 plasmids were UV irradiated (10J/m²). The cell-cycle profile of the transfected population was determined 48 hours later.