FIGURE 5.

Silencing of Cdh1 or Cdc20 by RNAi results in a cell cycle-dependent stabilization of Mps1 protein. A, Mps1 levels accumulate in G₁/S-synchronized cells transfected with Cdh1 siRNA compared with SCR control. 293T cells synchronized at G₁/S with a double thymidine block were treated with SCR or Cdh1 siRNA in the presence of thymidine keeping the cells at G₁/S for the duration of the experiment. Whole-cell extracts were Western-blotted with the indicated antibodies at indicated times post siRNA treatment. Cell cycle phases were monitored by flow cytometry. B, Mps1 degradation and progression into anaphase is delayed in Cdc20-depleted 293T cells. 293T cells transfected with SCR or Cdc20 siRNAs were incubated with nocodazole (Noc) for 16 h. Round, prometaphase-like cells were then collected by gentle shake off, replated into fresh medium, and collected at the indicated times for immunoblotting or FACS analysis. C, left panel, delay of mitotic exit in Cdc20-depleted cells. Results from flow cytometry show the percent of mitotic cells at the indicated times following nocodazole release. Right panel, graph of Mps1 protein levels (normalized to β-actin) in SCR and Cdc20 siRNA cells at indicated times following nocodazole release. Results shown are representative of at least three independent experiments.