JCS027730 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Determination of the amount of stable p21 (6Mycp21) that results in p21 levels similar to those observed after endogenous p21 upregulation. (A) U2OS cells were UV irradiated or treated with daunorubicin (D, 0.22 µM) and actinomycin D (A, 5 nM) for the indicated times. Whole cell extracts were collected and used for western blot analysis using p53, PIG3, Hdm2 and p21 antibodies. (B) U2OS cells were treated with daunorubicin (0.22 µM) and actinomycin D (5 nM) for 24 hours to trigger endogenous accumulation of p21 and cell-cycle arrest (data not shown). Different concentrations of 6Mycp21 plasmid were transfected and compared with the physiological levels of upregulated p21. The amount, 400 ng plasmid/1×10⁶ cells (shown in the figure), was selected and used in all the experiments described in this report. (C) The point mutation in 6Mycp21 (PCNA−) impairs p21 interaction both with endogenous and GFP-PCNA. U2OS cells were transfected with the indicated samples and lysed 24 hours after transfection. Immunoprecipitations were performed with anti-p21 polyclonal antibodies. Western blots of inmunoprecipitated samples and lysates were performed using PCNA, CDK2 and p21, and actin as loading control.

• Supplemental Figure S2 -

  Fig. S2. p21 and PCNA colocalize in foci that synthesize DNA when the CDK-binding domain of p21 is disrupted. (A) U2OS cells were transfected with GFP-PCNA and 6Mycp21 (CDK−). After PFA fixation, the colocalization between p21 and PCNA was analyzed by confocal microscopy, generating profiles of signal intensity along an arbitrary line drawn across the nuclei (left-hand panel). To determine BRDU/GFP-PCNA colocalization, U2OS were transfected with GFP-PCNA and 6Mycp21 (CDK−), and a short BrdU incorporation protocol was performed as in Fig. 1B. After methanol/acetone fixation, the profile of signal intensity was obtained as described for p21/PCNA colocalization (central panel). A similar procedure was used to analyze the colocalization between p21 and DNA synthesis foci (short pulse of BrdU incorporation with PFA fixation to avoid extraction of p21) (right-hand panel). (B) U2OS cells were transfected as in A. GFP-PCNA/p21 foci were evaluated by confocal microscopy. The position and size of single foci were schematized by analyzing a high-contrast 2D image (left). A 3D reconstruction was performed by integrating multiple z-stack images of the same nuclei (right). Arrows indicate that the larger foci observed with 6Mycp21 (CDK-) correspond to multiple individual foci in the 2D model.

• Supplemental Figure S3 -

  Fig. S3. Stable p21 does not impair early and late steps of NER. (A) U2OS cells were cotransfected with the indicated plasmids and GFP-PCNA as a marker for transfected cells. Samples were UV irradiated (80 J/m²) using polycarbonate filters. Thirty minutes after irradiation, cells were fixed and immunostaining was performed with XPB-specific antibodies. Single-cell results are reported in the upper set of panels. DAPI staining was used to visualize the nuclei. A quantification of three independent experiments showing the percentages of total cells that showed XPB recruitment to the irradiated spots in GFP-PCNA-positive cells is shown in the bar chart. Since proliferating cells were detected both in control (EV) and in 6Mycp21 (CDK−) transfected samples, images corresponding to diffuse and focal PCNA distribution are reported in both cases. (B) U2OS cells were transfected with the indicated plasmids and GFP-PCNA as a transfection marker. UV irradiation (20 J/m²), or not (non-irradiated, NI), and BrdU incorporation (100 µM) were performed as described in Fig. 2C. (C) Human fibroblasts obtained from healthy donors (XPA wt) and XPA-deficient fibroblasts were grown to confluence, UV irradiated and subjected to BrdU incorporation as described in Fig. 2C.

• Supplemental Figure S4 -

  Fig. S4. Effect of the different p21 constructs on the mobility of GFP-PCNA in living cells. (A) Representative example of the FLIP/FRAP (fluorescence lost in photobleaching/fluorescence recovery after photobleaching) experiments performed to determine the average time required to regain equivalent fluorescence intensity after photobleaching. U2OS cells were transfected with GFP-PCNA and half a nucleus was subjected to a bleaching laser pulse. Images were obtained in 5-second intervals and the fluorescence intensities in both the bleached and unbleached areas were measured using the Zeiss Time Series tool. The minimum time required for nuclei to reach equivalent levels of fluorescence intensity in both areas was determined and is shown in the right-hand panel. (B) The average time for convergence in fluorescence intensity was calculated in cells with (right) and without (left) GFP-PCNA foci. The standard deviation was calculated for the data obtained from at least five cells. The significance of the differences between the non-irradiated (NI) and UV-treated samples were assessed by Student's t-test for each p21 variant (***P<0.001, **P<0.01, *P<0.05). Full comparison was performed by one-way ANOVA with Tukey-Kramer post-test (tabulated data).

• Supplemental Figure S5 -

  Fig. S5. The p21-PCNA interaction allows p21 and PCNA focal colocalization both before and after UV irradiation but prevents pol η recruitment to those sites. (A) Merged panels from Fig. 1C and Fig. 3A showing PCNA and 6Mycp21 (CDK−) focal colocalization both before and after UV irradiation. (B) Merged panels from Fig. 4A showing that 6Mycp21 (CDK−) prevents pol η recruitment to focal stalled sites.

• Supplemental Figure S6 -

  Fig. S6. The p21-PCNA interaction promotes upregulation of γH2AX levels after UV irradiation. U2OS cells were transfected with the indicated p21 constructs and UV irradiated (10J/m²). Cells were fixed at the indicated time points and immunostaining with γH2AX- and p21-specific antibodies was performed. DAPI staining was used to visualize the nuclei.