Defective DNA repair in DT40 cCdc14A-KO and cCdc14B-KO cells. (A) The indicated cell lines were harvested either before (t = 0 h) or after IR, fixed, stained with PI and anti–γ-H2A.X, and analyzed by flow cytometry. (B) Quantification of the γ-H2A.X–positive cells in A (n = 3) is shown. (C) IB analysis of cCdc14A in cCdc14A-Res cells compared with WT. (D) WT, cCdc14A-KO, and cCdc14B-KO cells harvested either before (−IR 0 h) or 0.5 and 3 h after IR. Cells were fixed, stained for pH3 (red) and γ-H2A.X (green), and examined by fluorescence microscopy. Mitotic cells before and after IR. Anti–γ-H2A.X staining of the centrosome (arrowheads) was seen in some unirradiated cells. Bar, 5 µm. (E, left) The proportion of cells positive for pH3 and γ-H2A.X was scored as a percentage of total mitotic cells (n = 3; 100 mitotic cells per genotype). (right) The number of γ-H2A.X foci/cell was counted in projected and deconvolved images of mitotic cells positive for γ-H2A.X (n = 3; 20 mitotic cells). (F) The indicated cell lines were treated ± 1.5 µM DXR for 2 h and analyzed by single-cell gel electrophoresis (comet assay). Representative images are shown. Bar, 5 µm. (G) Tail moments for each time (n = 75; mean of two independent experiments) were quantified with ImageJ software. (H) Cell viability after IR analyzed by MTT assay (n = 3; P < 0.02). Error bars indicate mean ± SD.