Figure 5

Polo kinase 1 is required for CHK2 localization at centrosomes. (A) Inhibition of PLK1 activity with BI 2536 inhibits GFP-CHK2 association with centrosomes. U2OS GFP-CHK2 cells were incubated with doxycycline at 5 ng/ml for 32 h and treated for an additional 16 h with BI 2536 (100 nM) or monastrol (100 μM) in doxycycline-containing medium. Cells were then fixed and stained for centrosomes (red) and DNA (blue) and GFP-CHK2 was visualized by direct fluorescence (green). (B) Inhibition of PLK1 expression prevents GFP-CHK2 association with centrosomes during mitosis. U2OS GFP-CHK2 cells were transfected with a siRNA directed against PLK1 or control siRNA. 48 h post-transfection cells were fixed to examine GFP-CHK2 localization by fluorescence microscopy as in (A). (C) Western blot analysis of PLK1 expression shows a significant decrease in the expression of PLK1. Cell lysate from PLK1 siRNA-transfected U2OS cells was prepared from arrested mitotic cells collected by shake-off 48 h post-transfection. As a control, U2OS cells transfected with control siRNA were synchronized in mitosis by a 16 hours nocodazole block and protein extracts were prepared from the mitotic cells collected by shake-off.