FIG 5.

RNF126 is required for completion of NHEJ. (A) U2OS cells were transfected with RNF126 or control siRNAs, treated (or not treated [NT]) with bleomycin, incubated for the indicated times, and then subjected to a single-cell neutral gel electrophoresis (comet) assay of DSBs. Representative comet images and quantitative analysis of tail moments for 750 cells from three independent experiments are shown. Quantitative data are means ± SEM. *, P < 0.05 (Student's t test). (B) U2OS cells transfected with RNF126 or control siRNAs were exposed (or left unexposed) to IR (2 Gy) and subjected to immunofluorescence analysis of γH2AX (red) at 0.5 or 24 h after irradiation. Nuclei were stained with Hoechst 33342 (blue). Representative images and quantitative analysis of the total intensity of γH2AX foci in a total of 750 cells before or at 1, 3, 6, 12, 24, and 36 h after irradiation are shown. Quantitative data are means ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01 (Student's t test). (C) H1299dA3-1#1 cells expressing RNF126, Ku80, or control siRNAs were harvested 48 h after transfection with an expression vector for I-SceI [(+); (−), no transfection] for determination of the EGFP-positive cell fraction by flow cytometry. Quantitative data are means ± SEM from three independent experiments. **, P < 0.01; ***, P < 0.005 (Student's t test). The cells were also subjected to immunoblot analysis of the indicated proteins. (D) U2OS cells expressing RNF126 or control siRNAs were exposed to IR or UV-C radiation or treated with bleomycin before determination of colony-forming ability. Data are means ± SEM from three independent experiments. P values were <0.05 (*) and <0.01 (**) versus corresponding values for cells transfected with the control siRNA (Student's t test).