Fig. 2. Neither the PCNA-binding nor the CDK-binding domain of p21 inhibits early or late steps of NER. (A) U2OS cells transfected with the indicated plasmids were subjected to UV irradiation. Samples were collected at different time points and p21 protein levels were determined using specific antibodies. Actin was used as a loading control. (B) U2OS cells transfected with the indicated plasmids were UV irradiated (80 J/m²) using polycarbonate filters. Thirty minutes later, cells were fixed and immunostained with XPB- and p21-specific antibodies. DAPI staining was used to visualize the nucleus. Quantification is reported in the bar chart. The first column (EV) indicates the percentage of total cells that showed XPB recruitment to the irradiated spots. The other columns represent the percentage of p21-positive cells with XPB relocalization to irradiated spots. In all cases, at least 200 transfected nuclei/sample were counted. Values are the average and error bars are the standard deviation between equivalent samples in two independent experiments. (C) U2OS cells were transfected with the indicated plasmids and GFP-PCNA as a transfection marker. Samples were UV irradiated (20 J/m²) or not (non-irradiated, NI) and BrdU incorporation (100 µM) was performed for 4 hours. Cells were fixed and stained with BrdU-specific antibodies and DAPI. The white outline, generated using confocal software, was used to distinguish nuclear BrdU signal from mitochondrial DNA synthesis. The magnified (Zoom) images show the nuclei indicated by arrowheads. Quantification of the results obtained with all p21 mutants is reported in the bar chart (10 nuclei/sample were quantified). A complete panel showing all the p21 mutants is shown in supplementary material Fig. S3B.