FIG. 5.

The N-terminal-truncated FoxM1 mutant is insensitive to inactivation of cyclin A/cdk complexes. (A) 6xDB luciferase reporter transactivation by full-length FoxM1 or ΔN-FoxM1 in U2OS cells expressing DNcdk1 alone or together with cyclin A. (B) Transactivation of 6xDB by FoxM1 or ΔN-FoxM1 was measured in U2OS cells at 12 h after release from the G₁/S block. U2OS cells were either transfected with empty pSuper RNAi vector (1) or with an RNAi-expressing construct targeting cyclin A (2) or cyclin B (3). Endogenous cyclin A and cyclin B as well as ectopically expressed FoxM1 or ΔN-FoxM1 protein levels were viewed by Western blotting. *, nonspecific band. (C) Transactivation of 6xDB by the indicated constructs was measured in U2OS cells synchronized at the G₁/S transition by 24-h administration of thymidine (0 h) and at 12 h after release from the G₁/S block. Expression of all constructs was determined by Western blotting. (D) In vitro phosphorylation of the Flag-tagged wild type and 3A FoxM1 mutant. The flag-tagged wild-type and 3A proteins were transfected in 293T cells and immunoprecipitated using anti-flag antibody. The immunoprecipitates were then used in kinase assays with a radioactive label in the absence or presence of active purified cycA/cdk2 complex (Cell Signaling). Kinase activity was viewed by autoradiography (upper panel). Quantification of ³²P incorporation is shown (lower panel). (E) Quantification of the mitotic cell fraction in U2OS cells transfected with pS or pS-FoxM1 targeting vector in combination with RNAi-insensitive forms of the indicated proteins. The percentage of the mitotic cell population was measured using pH 3 staining 16 h after release from the G₁/S transition in the presence of nocodazole. Expression of all constructs was determined by Western blotting.