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Fig. 9. Bub1-deficient cells have a robust spindle checkpoint. (A) DLD-1 cells were transfected with control, Bub1 and BubR1 siRNA duplexes. 48 hours after transfection, the cells were fixed, stained with Hoechst and the number of cells in each phase of mitosis determined. (B) 48 hours after transfection, cells were incubated with nocodazole and the number of mitotic cells scored by fluorescence microscopy after 3 hours and 6 hours. At least 1000 cells were counted for each time point and values represent the mean and s.e.m. from at least three independent experiments. (C) HeLa cells were transfected with control, Bub1 and BubR1 siRNA duplexes and, 40 hours after transfection, cells were treated with nocodazole for 18 hours, at which point untreated cell lysates were analysed by western blotting to determine levels of protein repression. (D) The percentage of mitotic cells in the population was determined by MPM2 staining followed by FACS analysis and plotted as a bar graph. Values represent the mean plus s.d. of two independent experiments. (E) Phase images of asynchronous control, Bub1 and BubR1 repressed cells after the 18 hour nocodazole block (top). Cells were spun onto slides to detect DNA (red) and phospho-histone H3 (green) (bottom).
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