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. Author manuscript; available in PMC: 2010 Nov 1.
Published in final edited form as: Nat Chem Biol. 2010 Apr 11;6(5):359–368. doi: 10.1038/nchembio.345

Figure 2. Mps1-IN-1 and Mps1-IN-2 Induce Bypass of a Checkpoint-mediated Mitotic Arrest.

Figure 2

(a) Immunoblot of cyclin B from U2OS cells treated with Mps1 inhibitors. U2OS cells were arrested in mitosis by combination treatment of thymidine and nocodazole prior to treatment with nocodazole or co-administration with Mps1 inhibitor ± MG132 for 4 hrs. (b) Hela S3 cells expressing histone H2B-GFP were treated with nocodazole ± Mps1-IN-1. Cells were followed by immunofluorescence to determine exit from mitosis via nuclear envelope reassembly. 150 cells of each cell population were counted. (c) Selected frames from a time-lapse series of cells after treatment as in (b). Time is given in minutes:seconds. (d) Cumulative percentage of cells initiating anaphase. U2OS H2B–GFP cells were treated with DMSO or Mps1-IN-1 and imaged using fluorescence time-lapse microscopy. Time refers to the duration between nuclear envelope breakdown (NEBD) and anaphase initiation. 100 cells of each cell population were analyzed. (e) FACS analysis of UTRM10 cells expressing LAP-Mps1 WT or M602Q treated with Mps1-IN-1 or DMSO for 48 hrs. (f) Immunoblot showing induction of the RNAi-resistant LAP-tagged proteins. Clones were harvested and the relative levels of LAP-Mps1 in mitosis were compared to the UTRM10 parental cell line 17, 18. (g) Immunoblot of pHistone H3 in UTRM10 cells expressing LAP-Mps1 WT or M602Q after treatment with Mps1 inhibitors. UTRM10 cell lines arrested in mitosis by combination treatment of thymidine and taxol were treated with taxol ± inhibitor. All graphics were obtained from three independent experiments. Scale bars in C are 10 µm.