Immunodepletions of endogenous TOP2A were performed with protein Aconjugated magnetic beads

ere more explicit at 0.33 M nocodazole. Similar trends were observed with AURORA kinase inhibitors. Checkpoint overriding by reversine was not limited to HeLa Reversine is an MPS1 inhibitor Santaguida et al. 79 and Salmon, 2007). A consequence of the artificial stabilization of kinetochoremicrotubule attachment when the error correction pathway is inhibited is that the levels of checkpoint proteins at kinetochores are strongly reduced. To demon strate beyond reasonable doubt that the inhibition of MPS1 ATL-962 supplier causes a genuine checkpoint override rather than a mere satisfaction of the spindle checkpoint in the absence of error correction, as has been previously proposed for AURORA B inhibitors, we monitored the recruitment of the checkpoint proteins, an estab lished hallmark of checkpoint activity, to kinetochores at 3.3 M nocodazole. Even at 3.3 M nocodazole, both the RZZ and MAD1 were unable to localize to kinetochores. Thus, the disappearance of checkpoint proteins from kinetochores when MPS1 is inhibited is not caused by satisfaction of the spindle checkpoint by residual kinetochoremicrotubules in the absence of an error correction mechanism. Rather, this behavior reflects a genuine requirement of MPS1 in kinetochore recruitment of a sub set of checkpoint components. Reversine does not inhibit MEK1, nonmuscle Myosin II, or phosphatidyl-inositol 3-kinase in mitosis cells, as it was also observed with comparable potency in U2OS and retinal pigment epithelial cells. Reversine also caused an override of the checkpoint in the presence of Taxol, STLC, or the Plk1 inhibitor BI2536. MPS1 is required for localization of checkpoint proteins when microtubules are completely depolymerized Kinetochorebound microtubules contribute to removing the checkpoint proteins from kinetochores, Chen et al. identified NMMII, MEK1, and PI3K as putative targets of reversine. Although our characterization of reversine strongly supports inhibition of MPS1 as the main mechanism of reversine action in mitosis, we wished to test the possibility that NMMII, MEK1, or PI3K are targets of reversine during mitosis. The effects of blebbistatin, an NMMII inhibitor, were compared with the effects of reversine. At 100 M, blebbistatin did not cause any evident effects on chromosome align ment, suggesting that NMMII, the target of this inhibitor, does not contribute to chromosome alignment. Blebbistatin did not significantly affect the ability of mitotic HeLa cells to maintain a nocodazolemediated arrest. Because rever sine does not have obvious effects on cytokinesis until concentra tions of 25 M, at which concentrations we show that it inhibits Aurora B, we surmise that the mitotic phenotypes caused by submicromolar reversine are unlikely to be the result of the in hibition of NMMII and that if NMMII inhibition occurs, it does so at PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19833612 concentrations of reversine >25 M. To assess whether NMMII is a target of reversine at high concentration in mitotic cells, it will be necessary to sort out the relative effects of reversine on Aurora B and NMMII, as both of these proteins work in cytokinesis. We also compared the effects from adding MEK1 or PI3K inhibitors to the ability of HeLa cells to maintain a nocodazole mediated arrest. Neither the MEK inhibitor U0126 nor the PI3K inhibitor wortmannin affected the duration of the spindle check point in the presence of spindle poisons. Overall, these results indicate that NMMII, MEK1, and PI3K are not promi nent mitotic targets of reversine or el