(A) U2OS-CYCA-Luc cells were arrested in S-phase by growth in 0.2 mM mimosine. At 0, 3, 6, 9, and 12 h after removal of mimosine, cells were analyzed for DNA content by FACS after propidium iodide staining, or were lysed. (B, C) Cell extracts were analyzed by immunoblotting (B) or assayed for luciferase activity (C). (D, E) Immunoblot analysis of U2OS-CYCA-Luc cells (D), and luciferase activity in U2OS-CYCA-Luc cells or U2OS-Luc cells (E) transfected with Cdc20 or scrambled (negative control) siRNA. For normalization of luciferase or CYCA-Luc activity, the signal for untreated cells was set to 1. This experiment was repeated three times (n = 3). Error bars indicate standard error; *, p<0.05 compared with PBS.