Figure 1.

Plk1 is inhibited after the DNA damage checkpoint through regulation of T210 phosphorylation. (a) U2OS cells were synchronized in G2 by an 8 h thymidine release. After 8 h, cells were treated as indicated. Plk1 was immunprecipitated and samples were probed with the indicated antibodies. (b) Asynchronously growing U2OS cells expressing the Plk1 FRET-based biosensor were filmed and at time point 0 the indicated drugs were added. Cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) emission ratios were calculated and plotted over time. Stills show representative cells in each of the different conditions. Graphs depict analysis of 10 individual cells. Cells that entered mitosis before addition of the drugs are indicated in blue and cells that had active Plk1 at the time of drug addition are shown in red. Dashed gray lines indicate time point zero. (c) The Plk1 FRET-based biosensor was transiently transfected in cells stably expressing tetracycline-inducible Plk1-wt or Plk1-T210D. Expression was induced with tetracycline and individual cells were treated and analyzed as in b.