Figure 2.

GSK3 regulates the strength of the mitotic checkpoint. (A,B) HeLa cells exposed to 20 nM EPO with or without SB, ZM447439 (ZM), or CDK1 inhibitor RO3306 were tracked by phase contrast microscopy for mitotic entry and exit. (C–G) Asynchronous HeLa cells were tracked upon addition of Taxol or EPO by live cell imaging using phase contrast microscopy. GSK3 inhibitors, SB415286 or LiCl, or Aurora B inhibitor, ZM, was added and the live cell imaging continued to follow the same cells. The length of time before and after mitosis entry and mitotic exit was correlated. (as indicated in the box). (H) Hela cells were transfected with GSK3β RNAi or with scrambled siRNA. Cells were exposed to 1 μM Taxol 3-days post transfection and time lapse imaging used to analyze mitotic length. ~30 cells were analyzed per experiment. Western blot (WB) was done for GSK3β and Actin loading control, to show knockdown of GSK3β. siRNA against human GSK3β or scrambled siRNA was transfected into HeLa cells and 3 days post-transfection cells were subjected to WB analysis.