Fig. 8. Rereplication with reduced Cdk1 activity is induced when 293T cells that overexpress Cdt1 are arrested in M phase by nocodazole. (A) 293T cells were transfected with the indicated plasmids, selected with puromycin, and further treated with nocodazole. Whole cell lysates were then immunoblotted. (B) The DNA content of cells as in A was analyzed by flow cytometry and the percent of cells with rereplicated (>4N) DNA was calculated. The mean and standard deviation of two independent experiments is shown with representative raw data. (C,D) Suppression of ATM by shRNA reduces Chk2 phosphorylation and rereplication in Cdt1-overexpressing and nocodazole-treated 293T cells. The 293T cells were infected with retroviruses expressing ATM shRNA or control retroviruses and selected. Cells were then transfected with the indicated plasmids and treated with nocodazole. Whole cell lysates were then subjected to immunoblotting. The DNA contents of the cells were analyzed by flow cytometry and the percentage of cells with rereplicated (>4N) DNA was calculated. The mean and standard deviation of two independent experiments is shown for each experiment.