Figure 5
Rpp29 and Rpp21 are recruited to DNA damage sites in a PARP1-dependent manner. (A) Western blot analysis shows the efficiency of siRNA knockdown of PARP1. Protein extracts were prepared from mock and PARP1 siRNA-transfected U2OS cells and immunoblotted with PARP1 antibody. β-actin is used as a loading internal control. (B,C) Representative time-lapse images show subcellular distributions of EGFP-Rpp29 and EGFP-Rpp21 after laser microirradiation of mock and PARP1-depleted cells (Left). Line graphs (middle) depict fold increase in the relative fluorescence intensity of Rpp29 and Rpp21 at laser-microirradiated sites in mock and PARP1-siRNA transfected cells. Each measurement is representative of at least 10 cells. Error bars indicate SD. Column graphs (right) display the percentage of PARP1-depleted cells exhibiting accumulation of EGFP-Rpp29 and EGFP-Rpp21 at damage sites, as compared with mock-transfected cells. Error bars represent the SEM from two independent experiments. P-values were calculated by two-sided Student’s t-test relative to mock; ***p < 0.001. (D,E) Representative time-lapse images (left) show localization of EGFP-Rpp29 (D) and EGFP-Rpp21 (E) fusions to laser-microirradiated sites (marked by red arrow) in U2OS cells treated for 1 h with either DMSO or 5 μM of the PARP1/2 inhibitor (Ku- 0059436)(PARPi). Results shown are typical of two independent experiments and represent at least 30 different cells. Line graphs (middle) show fold increase in the relative fluorescence intensity of EGFP-Rpp29 and EGFP-Rpp21 at laser-microirradiated sites in untreated and PARPi-treated cells. Column graphs (right) display the percentage of PARPi-treated cells with accumulation of EGFP-Rpp29 and EGFP-Rpp21 at damage sites, as compared with untreated cells. Error bars express the SEM from two independent experiments. P-values were calculated by two-sided Student’s t-test relative to DMSO; ***p < 0.001.
