ATAD5 knockdown delays S-phase cell cycle progression. In all experiments, HeLa cells were analyzed 72 h after transfection of ATAD5 or control siRNAs unless otherwise specified. (A) Cell cycle was measured by flow cytometry. The relative percentage of cell cycle stages was calculated from three independent experiments and displayed as a graph with the mean value below. (B) 48 h after transfection, cells were arrested with a double thymidine block. At indicated times after release from the G1/S block, cells were pulse-labeled with BrdU for 30 min and collected for cell cycle analysis. The data shown are from a single representative experiment out of two repeats. Numbers are the relative percentage of cell cycle stage. Early and late S phases indicate 7-AAD low and high cells, respectively. (C) Cell numbers were colorimetrically measured. **, P < 0.05; *, P < 0.01, by t test. Error bars indicate SD. (D) Cells were pulse-labeled with BrdU for 10 min, fixed, stained with anti-BrdU antibody, mounted with DAPI reagent, and analyzed by confocal microscopy. Relative nuclear surface size (n = 50 in each condition from a single experiment) was calculated. Red bars in the graph, median value; P, significance by t test. (E) Cells were treated with 20 gray (Gy) of γ-radiation, incubated for 30 min, fixed, and stained with both anti-PCNA and anti-γH2AX antibodies and analyzed by confocal microscopy. Bar, 5 µm. (F) Cells were treated with 4 µM CPT for 1 h and total extracts were isolated for immunoblot assay.