KMN is required for the spindle checkpoint in human cells. (A) Flow cytometry (FACS) analysis of HeLa cells transfected with the indicated siRNAs and treated with nocodazole. The mitotic indices (defined as the percentage of cells with 4C DNA content and high MPM2 staining) are indicated. (B) Quantification of mitotic indices of cells in A, with means and SD (error bars) shown (n = 3). (C) Lysates of cells in A and B were blotted with the indicated antibodies. (D and E) Quantification of mitotic indices of HeLa cells transfected with indicated vectors and siRNAs and treated with nocodazole (mean ± SD [error bars], n = 3). (F) Quantification of mitotic indices of HeLa cells stably expressing Dsn1-WT-GFP that were mock transfected or transfected with siMis12C in the absence (–) or presence (+) of doxycycline (Dox) and treated with nocodazole (mean ± SD [error bars], n = 3). (G) Lysates of cells in F were blotted with the indicated antibodies. Asterisks indicate nonspecific bands. (H) Mitotic HeLa cells transfected with the indicated siRNAs were stained with the indicated antibodies and DAPI. Selected channels (as labeled by the corresponding colors) were included in merged images. Boxed regions were magnified and shown in the rightmost column. Normalized intensities (mean ± SD [error bars], n = 400) of kinetochore staining are shown. Bars, 5 µm (1 µm in magnified images on the right).