RAD51C promotes cell cycle arrest in response to DNA damage. (A) RAD51C depletion reduces IR-induced S-phase delay. U2OS cells were irradiated (1 Gy) 48 h after transfection with control or RAD51C siRNA and, 2 h later, fixed for propidium iodide staining and flow cytometry. G1, S, and G2/M populations were assigned using CellQuest. (B) Analysis of the G2/M checkpoint. As in A, but cells were stained with an antibody against phosphorylated histone H3 and propidium iodide. (A and B) The mean and standard deviation from three independent experiments are shown. Significance in difference between the grouped values was tested using an unpaired t test. (C) shRNA-mediated reduction of RAD51C expression in primary MEFs. Cells were infected twice at 12-h intervals with retroviruses expressing control or RAD51C shRNA and then selected with puromycin for 48 h. Cell extracts prepared 3 d after the first infection were analyzed by Western blotting against mouse RAD51C and tubulin. (D) Cells treated as in C were incubated with colcemid for 4 h and processed for FISH analysis of metaphase chromosome spreads with a Cy3-conjugated telomeric peptide nucleic acid probe (red). Arrowheads point to chromatid breaks. (E) Quantification of chromatid and chromosome break frequencies in metaphase spreads from MEFs established from two littermate embryos, each transfected with either control or RAD51C shRNA. 30–50 metaphases were analyzed for each sample. Bar, 10 µm.