Fig. 1. FOXM1 is phosphorylated and translocated to the nucleus before entry into the G2/M phase. (A) Flow diagrams of asynchronized and synchronized BJ1 cells. BJ1 cells were synchronized at the G1/S boundary by serum starvation/aphidicolin double block. Cells were released from arrest by removal of aphidicolin. At different time intervals after release, cells were harvested and stained with propidium iodide for DNA analysis using a flow cytometer. Synchronized cells gradually progressed through the cell cycle and G2/M cells could be detected at both 9 hours and 12 hours after release. (B) Cells at different time intervals after aphidicolin release were harvested for immunoblot analysis using anti-FOXM1 and anti-tubulin antibodies. Progressive mobility shifts (denoted by arrowheads) of the FOXM1 band were observed at 9 hours and 12 hours after release. (C) The cell lysate at 9 hours after release was treated with calf intestine phosphatase (for 1 hour) before immunoblot analysis. Such treatment abolished the mobility up-shift of the FOXM1 band. (D) BJ1 cells grown on coverslips were synchronized by serum starvation/aphidicolin double block and fixed at various time points after removal of aphidicolin. Cells were immunostained with anti-FOXM1 antibody and counterstained with propidium iodide to detect nuclear DNA. Merged images of FOXM1 and nuclear staining are also shown. FOXM1 was predominantly cytoplasmic (arrows) at 0 hour, 3 hours and 6 hours after release; FOXM1 became mainly nuclear (arrowheads) at 9 hours and 12 hours after release.