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Fig. 3. The p21-PCNA interaction impairs the assembly of new GFP-PCNA foci after UV irradiation. (A) U2OS cells transfected with GFP-PCNA and the indicated p21 plasmids were UV irradiated (20 J/m²). Six hours later, cells were fixed and the sub-nuclear distribution of PCNA and p21 determined by confocal microscopy. Non-irradiated (NI) controls obtained in parallel are shown in Fig. 1C. After UV irradiation, PCNA foci were similar to replication PCNA foci (EV, left-hand panel) or much smaller (EV, right-hand panel). When the 6Mycp21 (CDK–) mutant was transfected, the PCNA distribution ranged from diffuse to increasingly collapsed nuclear foci as shown in the set of panels to the right. Merged panels are shown in supplementary material Fig. S5. (B) The percentage of cells with GFP-PCNA foci before and after 6 hours of UV irradiation (20 J/m²) was determined. In all cases, at least 200 transfected nuclei were counted. Values are the average and error bars are the standard deviation between equivalent samples in three independent experiments. The significance of the differences between the NI and UV samples was assessed by Student's t-test for each p21 variant (^***P<0.001; ns=P>0.05, not significant). Further statistical comparison was performed by one-way ANOVA with Tukey-Kramer post-test (Table 1). (C) The percentage of cells with GFP-PCNA foci was determined at different time points after UV irradiation (20 J/m²). In all cases, 200 transfected nuclei/sample were counted. Values are the average and error bars are the standard deviation between equivalent samples in two independent experiments.

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