Enforced expression of Skp2 abrogates antiestrogen-mediated cell cycle arrest in MCF-7 cells. (a) MCF-7 breast cancer cells were synchronized in G1 phase by treatment with 1 μM tamoxifen for the indicated times (lanes 1–3). The cells were then restimulated to enter the cell cycle by addition of 500 nM estradiol for the indicated times (lanes 4–6). Cell extracts were analyzed by immunoblotting with Ab’s to the indicated proteins (top four panels) and for Cdk2 kinase activity assayed using histone H1 (HH1) as a substrate after immunoprecipitation of Cdk2 (bottom panel). (b) MCF-7 cells infected with retroviruses expressing the indicated proteins were either left asynchronous or were synchronized in G1 by depletion of estradiol and treated with tamoxifen for 48 or 96 hours, as indicated. The percentage of MCF-7 cells in S-phase was then analyzed by BrdU incorporation. The results shown in the first 10 bars are the mean percentage obtained from at least three independent experiments.