FIG. 7.

Coexpression of functional CDK4 and cyclin D1 proteins rescues FoxO-induced G₁ arrest in NIH 3T3 cells. NIH 3T3 fibroblasts were transfected with empty vector (control) or the indicated expression plasmids and cotransfected with limiting amounts of a vector encoding the surface marker CD20. Thirty-two hours later, cells were treated for 16 h with nocodazole to trap cells in G₂-M and stained with propidium iodide. DNA profiles of CD20-positive cells were recorded by FACS analysis. (A) Induction of G₁ arrest by FoxO3a.A3 expression. As a positive control for G₁ arrest, cells were transfected with an expression plasmid for the CDK inhibitor p21^Cip1/Waf1. (B) Effect of cyclin D1 (Cyc D1)/wild-type CDK4 (CDK4) or Cyc D1/kinase dead CDK4 (CDK4KD) cotransfection on FoxO3a.A3-induced G₁ arrest. The bars indicate percent increases in G₁ cells + standard deviation compared to the ratio of G₁ cells after transfection with an empty control vector. Data are obtained from three independent experiments. Expression of exogenous HA-CDK4 and HA-CDK4KD was confirmed by Western blotting using a monoclonal anti-HA antibody (right).