Chemical Genetics of Mps1 Figure 5. Mps1 inhibition accelerates mitosis. A–C: Time-lapse analysis by DIC microscopy of mitotic progression of HCT-derived (A, B) and UTR- derived (C) cells treated with DMSO or 23dMB-PP1. Images in A display morphology of HCT-derived cells imaged at the indicated timepoints after NEB (nuclear envelope breakdown). Graphs in B and C show cumulative percentages of cells that display first signs of furrowing at indicated times after NEB. Dashed lines indicate half-time to furrowing. doi:10.1371/journal.pone.0010251.g005 pS10-H3 levels in HeLa cells [12]. Since one difference between these experiments is whether or not cells entered mitosis with intact Mps1 activity, we next examined pS10-H3 levels in cells that had seen 23dMB-PP1 prior to mitotic entry. For this, cells were treated with DMSO or 23dMB-PP1 for 1 hour, after which taxol/ MG132 was added for an additional 2 hours. Analysis of these mitotic samples showed significant reduction of pS10-H3 levels in 23dMB-PP1-treated Mps1as cells but not Mps1WT cells (Fig. 6E, F and S5). The magnitude of this decrease is less prominent than direct inhibition of Aurora B activity using ZM447439 (Fig. 6E, F and S5) [33], which is in line with our previous observations that showed that while Mps1 could enhance Aurora B activity, it was not absolutely critical [12]. Selective, short-term inhibition of Mps1 kills cells Long-term Mps1 RNAi kills U2OS, HCT116 and LS174T cells [16,17] and Mps1 inhibition has been proposed as an effective way to induce a lethal dose of chromosome segregation errors [17,38,39]. To examine if selective and penetrant inhibition of the catalytic activity of Mps1 is lethal, UTR- and HCT-derived Mps1wt and Mps1as cells were treated with 23dMB-PP1 for 8 days after which colony outgrowth was scored. 23dMB-PP1 had no off-target effect on viability during this period, as Mps1wt cells remained fully viable despite prolonged exposure to 23dMB-PP1 (Fig. 7A). As shown in Fig. 7B, however, no Mps1as cells survived prolonged Mps1 inhibition. To approach a more relevant duration of inhibition in potential future clinical settings, we next tested the effect of short-term inhibition of Mps1 on cell viability. Addition of 23dMB-PP1 for 2 days and subsequent removal of the inhibitor until day 8 left no (HCT-Mps1as) or very few (UTR-Mps1as) cells alive (Fig. 7B). These data show that efficient inhibition of Mps1 for a relatively short period of time is sufficient to kill cells. PLoS ONE | www.plosone.org 7 April 2010 | Volume 5 | Issue 4 | e10251