Chemical Genetics of Mps1 Figure 3. Mps1 kinase activity promotes localization of Mad1, Mad2, Bub1 and Cdc20 to unattached kinetochores. A, B: Immunolocalization of Mad1, Mad2, Bub1, BubR1, CENP-E, Cdc20, and centromeres (ACA) in HCT-derived (A) or UTR-derived (B) cells treated with nocodazole and MG132 for 1 hour in combination with DMSO or 23dMB-PP1. Graphs represent quantifications of kinetochore signal intensities for the investigated proteins at all kinetochores in a single cell as a ratio of the ACA signal. Data are average of three experiments, 10 cells per experiment (+/2 SD). Representative images are shown. doi:10.1371/journal.pone.0010251.g003 was measured. Half of DMSO-treated HCT-Mps1WT and HCT- Mps1as cells had proceeded to furrow ingression 26 and 22 minutes after NEB, respectively (Fig. 5B). Strikingly, whereas addition of 23dMB-PP1 had no effect on the time to furrow ingression of HCT-Mps1WT cells, half the 23dMB-PP1-treated HCT-Mps1as cells showed signs of furrow ingression in as little as 10 minutes and no cell took longer than 20 minutes to reach this furrowing stage of mitosis (Fig. 5A, B). Further evidence for the high penetrance of Mps1 inhibition in these cells was illustrated by the fact that exit from mitosis in taxol was within a similar PLoS ONE | www.plosone.org 5 April 2010 | Volume 5 | Issue 4 | e10251