FIG. 3.

Cyclin A is required for FoxM1 target gene expression and for proper G₂/M progression. (A) Western blot analysis of endogenous FoxM1 protein in human U2OS cells transfected with empty pSuper or two different pS-cycA-targeting constructs. Cells were blocked at the G₁/S transition by 24-h administration of thymidine and released from the thymidine block for 12 h or 14 h in the presence of nocodazole. (B) U2OS cells cotransfected with spectrin-green fluorescent protein (GFP) plasmid and empty pSuper or pS-cycA targeting constructs were synchronized at G₁/S transition by thymidine treatment and released from the block for 14 h in the absence (left graphs) or presence of nocodazole (right graph). The left graphs show FACS analysis of DNA profiles of spectrin-GFP-positive cells using propidium iodide staining. The right panel shows the quantification of the mitotic cell population in nocodazole-treated cells using phospho-histone H3 staining (pH 3). (C) Western blot analysis of FoxM1 target gene (CENP-F and cyclin B1) expression in U2OS cells expressing pS or pS-cycA targeting vectors blocked at the G₁/S transition by 24-h administration of thymidine and released from the thymidine block for 14 h in the presence or absence of nocodazole (left panel). RT-PCR analysis of CENP-F and cyclin B1 mRNA levels in U2OS cells transfected with control siRNA or siRNA oligonucleotides targeting cyclin A and released from a thymidine block for 12 h (right panel). (D) Western blot analysis of FoxM1 target gene expression in human U2OS cells that were either transfected with empty pSuper RNAi vector (pS), with an RNAi-expressing construct targeting FoxM1 (pS-FoxM1), or with an RNAi-expressing construct targeting cyclin A (pS-cycA) or were cotransfected with both pS-FoxM1 and pS-cyclin A targeting constructs.