Figure 4.

Depletion of Cdh1 leads to unscheduled activation of Chk-1 activity in the G1 phase. (A) Depletion of Cdh1 by siRNA treatments leads to a significant induction of Claspin protein and activation of the Chk1/p53 pathway in asynchronized U2OS cells. (B) HeLa cells were transfected with indicated siRNA oligos, and 6 h after transfection, cells were synchronized with nocodazole. Eighteen hours later, cells were released back into the G1 phase, and at the various indicated time points, cells were lysed for immunoblot analysis using the indicated antibodies. (C) HeLa cells were transfected with the indicated siRNA oligos; 6 h after transfection, cells were synchronized with nocodazole. Eighteen hours later, cells were released back into the G1 phase. At various indicated time points, cells were pulsed with BrdU for 30 min, and afterward immunohistochemistry staining was performed with anti-BrdU antibody to detect the percentage of BrdU-positive cells. (D) Illustration of the proposed model by which Cdh1 could potentially activate Chk1 and p53 activity by controlling Claspin destruction.