Cdc14B phosphatase activity is required to prevent HU-induced centriole amplification. (A, top) Cdc14B-GFP fusion proteins were induced by 4 μg/ml DOX in the presence of 2 mM HU for 72 h in U2OS Tet-On stable cell lines carrying different Cdc14B-GFP constructs as indicated. Centrioles were visualized by anti-centrin staining (red) and overlaid with Cdc14B-GFP (green) and DAPI (blue). Note that Cdc14B^C314S-GFP was not detectable at centrioles (arrow). Insets show magnified images of centrioles. Bar, 5 μm. (bottom) The percentage of cells with more than four centrioles was calculated from both induced (+DOX) and uninduced (−DOX) Cdc14B-GFP stable clones as indicated. Data shown represent the means ± SD of three independent experiments from two individual Cdc14B-GFP stable clones. At least 500 cells were counted in each experiment. (B, top) U2OS Tet-On cells were transfected as indicated. 16 h after transfection, cells were incubated with (+HU) or without (−HU) 2 mM HU and 4 μg/ml DOX for 72 h. Centrosomes were visualized by γ-tubulin staining (red). Representative centrosome amplification was detected in mock-transfected cells after HU treatment but not in pBI-tet-Cdc14B^WT-GFP transfected cells where Cdc14B^WT-GFP (green) associated with centrosomes. Insets show magnified images of centrosomes. DAPI (blue), DNA. Bar, 5 μm. (bottom) The percentage of cells with the indicated centrosome numbers was calculated from the experiments shown in the top panel. Centrosomes were counted in both mock and Cdc14B-GFP–transfected cells (Cdc14B-GFP–positive at centrosomes). All the data are shown as the means ± SD of three independent experiments. At least 500 cells were counted in each experiment. (C) Representative fluorescence-activated cell sorting profile on cell cycle distribution of DOX-inducible Cdc14B-GFP U2OS Tet-On stable clones. Cells were cultivated in the presence or absence of 4 μg/ml DOX for 72 h. Positions of cells with 2 N and 4 N DNA contents are labeled with arrowheads.