Fig. 2.

Chk1 depletion causes mitotic arrest. (A) HeLa cells were transfected with siRNA targeting site 454 of Chk1. At 2 d after transfection, cells were treated with 200 ng/ml nocodazole (Noc.) for 10 h and harvested for Western blotting using the antibodies indicated. (B) HeLa cells growing on coverslips were transfected with siRNA targeting site 454 of Chk1, harvested at 1 or 2 d after transfection, and stained with phospho-H3 antibody. (C) Chk1 was depleted with siRNA in well-synchronized HeLa cells by using the double thymidine block as described in the text. Ten hours after release from the block, cells were harvested and analyzed by Western blotting using the antibodies indicated. (D) At different times after release from the double thymidine block, cells were analyzed by anti-phospho-H3 immunofluorescence staining. (E) Histograms quantifying the results from D indicate the formation of cells with multiple or fragmented nuclei after Chk1 depletion.