Mad2, our data further indicate that Mps1 is essential for recruitment of Cdc20 as well as Bub1 to unattached kinetochores, in agreement with some [11,25] but not other [12,24] previous reports using Mps1 RNAi or Mps1 protein depletion. Mps1 inactivation affects the quality of chromosome segregation in unperturbed mitosis [16,21]. Using time-lapse microscopy we show that inactivation of Mps1 accelerates mitosis. A similar phenotype was reported for cells depleted of Mad2 or BubR1 [40]. The regulation of mitotic timing by Mad2 and BubR1 is kinetochore-independent, since Mad1 depletion, while disabling the mitotic checkpoint, did not affect normal mitotic timing [40]. It is therefore unlikely that lack of recruitment of Mad1 and Mad2 to kinetochores in Mps1-inhibited cells contributes to accelerated mitosis. An explanation for the timing phenotype, proposed by Meraldi et al., and in agreement with experimental data [41], is the possibility that sufficient MCC exists in interphase to constrain APC/C activity long enough to allow full chromosome biorienta- tion in the majority of cells. In this model, only presence of persistent unattached kinetochores requires additional MCC formation to further prolong mitosis. Given the timing phenotype of Mps1-inhibited cells, it is therefore possible that Mps1, in addition to influencing Mad1-dependent MCC formation, affects stability or inhibitory potential of pre-assembled MCC. Further studies are needed to examine this hypothesis. Due to its central role in mitosis, Mps1 is an attractive target for anti-cancer therapies. A recent study from our lab proposed that elevating the frequency of chromosome segregation errors by partial Mps1 reduction can be of clinical relevance, especially with other treatments that affect fidelity of chromosome segregation [17]. We show here for the first time that specific inhibition of Mps1 enzymatic activity efficiently kills human cells. Moreover, a short pulse of inhibition followed by recovery caused lethality in a significant fraction of cells. As such a pulse of Mps1 inhibition is more relevant to potential future clinical situations than potent, long-term kinase inhibition, these data illustrate that specific and penetrant inhibition of Mps1 activity might indeed be a promising anti-cancer strategy. Materials and Methods Tissue Culture and Treatments UTR-Mps1WT, UTR-Mps1as, HCT-Mps1WT and HCT- Mps1as cells were derived from UTRM10 [11,16,17] and HCT- TRM (named HCT-116-TetRMps1 clone#2 in Ref. [17]) cell lines, respectively. Briefly, these cell lines stably express TetR and carry pSuperior-retro-puro-Mps1, resulting in depletion of Mps1 protein upon addition of doxycycline. To generate the derivative cell lines that express Mps1WT and Mps1as, UTRM10 and HCT- TRM cells were transfected using the calcium phosphate method with the different LAP-Mps1 alleles and selected with doxycycline (1 mg/ml). Single colonies were selected after limiting dilution. HCT-TRM-derivative cells were grown in RPMI (Lonza) with 6% Tet-approved FCS (Clontech), supplemented with pen/strep (Invitrogen). UTRM10-derivative cells were grown in DMEM (Lonza) with 8% Tet-approved FCS (Clontech), supplemented with pen/strep (Invitrogen) and Ultra-Glutamine (Lonza). Thymidine (2.5 mM), nocodazole (660 nM), taxol (1 mM), MG132 (10 mM), monastrol (200 mM), STLC (10 mM), doxycy- cline and puromycin (1 mgNml21) were all from Sigma, ZM447449 (Tocris Bioscience) was added at 2 mM and 23dMB-PP1 at 1 mM. Chemical Genetics of Mps1 (Upstate), pT676-Mps1 [16], BubR1-300A (Bethyl), Mad2 (custom polyclonal Rb antibody. Briefly, His6-Mad2 (pQE80L-hMad2) was expressed in BL21 cells, purified using Ni-agarose (Qiagen) and injected into New Zealand rabbits (Covance). Mad2 antibody was affinity purified from rabbit serum using His6-Mad2), Bub1 (Abcam), Mad1 (SantaCruz Biotechnology), a-tubulin (Sigma), CREST (Cortex Biochem), MPM2 (Upstate), Cdc20 (SantaCruz Biotechnol- ogy), Bub3 (BD Trans Lab) and Borealin (a gift of W. Earnshaw). Secondary antibodies included anti-human Alexafluor647 and anti- rabbit Alexafluor488 (Molecular Probes) for immunofluorescence studies, anti-mouse-Cy5 (Jackson) for FACS and anti-Mouse/Rabbit Alexa680/800 (Molecular Probes) for immunoblotting. Flow cytometry HCT- and UTR-derived cell lines were released from a 24 hours thymidine block into nocodazole or 23dMB-PP1 or a combination of both for 18 hours, harvested and fixed in 70% ice- cold ethanol for 24 hours, and analyzed using MPM2 immuno- stains as described [42]. Immunofluorescence and live cell imaging For immunolocalization studies, cells were plated on 12 mm coverslips, pre-extracted with 0.2% TritonX-100 in warm PEM (100 mM PIPES pH6.8, 1mM MgCl2 and 5mM EGTA) for 1 minute, fixed with 3% PFA in PBS, blocked with 3% BSA in PBS for 1 hour, incubated with primary antibody for 16 hrs at 4uC, washed with PBS/0.1% TritonX-100 and incubated with secondary antibodies for an additional 1 hour at room tempera- ture. Coverslips were washed and submerged in PBS containing DAPI, washed again and mounted using ProLong Antifade (Molecular Probes). All images were acquired on a DeltaVision RT system (Applied Precision) with a 100X/1.40NA UPlanSApo objective (Olympus) using SoftWorx software. Images are maximum intensity projections of deconvolved stacks. For quantification, all images of similarly stained experiments and acquired with identical illumination-settings were analyzed using ImageJ. Average pixel intensities of the total of all regions encompassing centromeres plus kinetochores were determined in the various channels and corrected for background. For live cell imaging, cells were plated in 4-well chambered glass-bottom slides (LabTekII) or 24-well glass bottom plates (MatTek), transfected and imaged in a heated chamber (37uC and 5% CO2) using a 20X/0.5NA UPLFLN objective on a Olympus IX-81 microscope, controlled by Cell-M software (Olympus). 16 bits DIC (5 msec exposure) images were acquired every 2 minutes using a Hamamatsu ORCA-ER camera. Images were processed using Cell-M software. Immunoprecipitation HCT-Mps1as cells were released from a 24 hours thymidine block into nocodazole for 18 hours, harvested and lysed in 50 mM Tris 7.5, 150 mM NaCl, 1% TX-100, 2 mM MgCl2, 5 mM EDTA, supplemented with protease inhibitors and phosphatase inhibitors for 10 minutes on ice. The cleared extract was incubated with 10% protein A-agarose beads (Roche)/antibody mix for 2 hours at 4uC on a rotating wheel. The beads were washed twice with lysis buffer. Supernatant and beads were processed for SDS-PAGE and the proteins were transferred to nitrocellulose membranes for immunoblotting. Antibodies The following primary antibodies were used for immunoblotting, immunofluorescence imaging and FACS analyses: Mps1-NT Colony Formation Assays Cells (50,000/well) were plated in 6-well plates (Costar). Cells were grown in media supplemented with DMSO or 23dMB-PP1 PLoS ONE | www.plosone.org 10 April 2010 | Volume 5 | Issue 4 | e10251