Figure 7.

Two semi-redundant mechanisms attach KMN to mitotic kinetochores. (A) Two proposed mechanisms of KMN assembly at kinetochores. In cells with depolymerized microtubules, inactivation of both is required to reduce the KMN level at kinetochores below that needed for checkpoint signaling. (B) Quantification of mitotic indices of Dsn1-WT/EE-GFP-expressing HeLa cells transfected with siCENP-T and treated with nocodazole and ZM (mean ± SD [error bars], n = 3). (C and D) Dsn1-WT/EE-GFP-expressing HeLa cells were transfected with siDsn1 and siCENP-T, arrested in mitosis by nocodazole, and further incubated with MG132 (NM) or with both MG132 and ZM (NM+Z). Mitotic cells were stained with DAPI and the indicated antibodies. The boxed regions of merged images are magnified and shown in the rightmost column. Bars, 5 µm (1 µm for magnified images). (E) Quantification of the relative kinetochore intensity of Ndc80 in C and D (mean ± SD [error bars], n = 400).