Fig. 4.

BRC8-2 impairs RAD51 foci formation in human U2OS cells. (A) Representative images of U2OS cells expressing GFP-NLS, that is GFP with a nuclear location signal only (GFP), a GFP-NLS-BRC8-2 peptide (GFP-BRC8-2), or a GFP-NLS-BRC4 peptide (GFP-BRC4). Cells were monitored 3 h after no treatment (No IR) or irradiation with 3 Gy (3Gy 3h) for GFP fluorescence or stained with RAD51 or DAPI as indicated. (B) Dot plot graph from one biological replicate plotting the number of RAD51 foci per GFP-positive cell. Mean values for each population are indicated with a bar. More than 50 GFP-positive cells were analyzed for each condition. Statistical analysis was done using Kruskal–Wallis rank sum test followed by Dunn’s procedure for pairwise comparison (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant). (C) Bar graph showing the average of the mean RAD51 foci per GFP-positive cell from four independent biological experiments. Data are presented as mean ± SEM, n = 4 biological repeats. **P < 0.01, ***P < 0.001; ns, not significant, using ANOVA test [F(5,15) = 15.29, P < 0.0001], assuming sphericity) followed by Tukey’s method. (D) Representative cell cycle profiles from GFP-positive cells transfected with GFP-NLS (GFP) or GFP-NLS-BRC4 (GFP-BRC4) or GFP-NLS-BRC8-2 (GFP-BRC8-2). Cells were analyzed by flow cytometry 3 h after no treatment (No IR) or irradiation with 3 Gy (3Gy 3h). PI, propidium iodide. (E) Bar graph showing the percentage of cells in G1 phase. Data are the mean values from three independent biological experiments ± SD.