Fig. 1: sPIR^p21 is as efficient as full-length p21 in impairing TLS-associated events after UV treatment.

A Schematic presentation of the sPIR^p21 variants used in this study encoding amino acid residues 139 to 164 of p21, fused to a 6-myc-tag at the N-terminus. The eight amino acid sequence corresponding to the PIP box are within the blue box. The bipartite NLS is represented by the dark red box. To generate the sPIR^p21ΔP, three point mutations in the sPIR^p21 sequence were introduced (M147A, D149A, F150A) to disrupt the interaction of p21 with PCNA [23]. NLS: nuclear localization signal. For further details about the mutants used, see the “Materials and methods” section. B Western blot of lysates obtained from U2OS cells transduced with sp21, sPIR^p21 or sPIR^p21ΔP lentiviral vector treated with UV (40 J/m²) 48 h after transduction. Whole cell extracts were prepared at the indicated time points. Anti-myc antibodies were used to detect myc-tagged p21. Actin was used as a loading control. A representative experiment is shown. N = 2 (N number of independent experiments). C Representative images of nuclei transfected with GFP-Pols under untreated (NT) and UV irradiated (UV) conditions. D Percentage (mean ± SD) of U2OS cells with detectable nuclear foci of the indicated DNA polymerase. Samples were transfected with the indicated GFP-Pols and 5 h later transduced with EV, sp21, sPIR^p21 or sPIR^p21ΔP. 48 h later, cells were treated with UV (40 J/m²) and fixed 4 h later. Transduced cells were detected by using an anti-myc specific antibody, and the focal organization of GFP-Pols was quantified in 200 nuclei positive for both GFP and myc. N = 3 (Statistics: one-way ANOVA, Tukey post test). The letters on top of the columns in this graph, as well as in all other graphs in this study with statistical analysis, mark grouped samples that are not significantly different. Samples not sharing a letter are significantly different. For more information about statistical analysis, see “Materials and methods”. E Labeling protocol used for the DNA fiber spreading assay. Bottom: representative image of a single bicolor DNA fiber of U2OS cells. F Quantification of IdU track length from U2OS cells transduced with the indicated vectors. Median is shown in black. Cells were either mock (NT) treated or treated with UV (40 J/m²). ̴200 fibers/sample were analyzed. Data shown for the condition NT EV is shown again in Fig. 2I. N = 2 (Statistics: Kruskal–Wallis, Dunn post test). G Western blot of U2OS cells transduced with the indicated vectors. 48 h after transduction with the indicated vectors, cells were either mock treated (NT) or treated with UV (40 J/m²). 4 h later samples were harvested for whole cell extraction. PCNA ubiquitination and PCNA were detected using specific antibodies against ubiquitinated PCNA and PCNA respectively. Ku70 was used as a loading control. A representative experiment is shown. N = 2.