Fig. 4. Bub1 and aurora kinase activity cooperate to maintain the spindle checkpoint. (A) DLD-1 cells were transfected with control or Bub1 siRNA duplexes, and then exposed to spindle toxins, plus or minus ZM447439. Cells were then fixed, the DNA stained, and the mitotic index determined by microscopy analysis using chromosome condensation as a visual marker. Whereas control cells exposed to nocodazole plus ZM447439 () accumulate efficiently in mitosis, Bub1 repressed cells () do not. (B) HeLa cells were transfected with control or Bub1 siRNA duplexes, synchronised in mitosis by selective detachment following a 12 hour nocodazole block, and then released into spindle toxins plus or minus ZM447439. Cells were then reharvested, centrifuged onto glass slides, fixed, the DNA stained, and the mitotic index determined by microscopy analysis. Relative to the controls, Bub1-repressed cells exit mitosis faster in nocodazole plus ZM447439. Values represent means±s.e.m. from three independent experiments.