Figure 4.

A, the percentages of cells at each stage of the cell cycle were analyzed by flow cytometry after PI staining. Bub1b-knockdown cells showed a decrease in G1 phase cells and an increase in >4N DNA content in both Rh30 and RD cell lines at day 5 after treatment with doxycycline to induce shRNA constructs. Data are mean ± SE. The results were representative of three independent experiments. B, chromosome numbers were quantified in Bub1b knockdown or control cells (n=50). C, Live-cell time-lapse microscopy studies demonstrated that knockdown of Bub1b promotes endoreduplication and mitotic catastrophe. Cells were treated with doxycycline for 4 days to induce the siRNA constructs, and then collected mitotic cells by shake-off, added these cells to a 6-well plate at very low density, incubated overnight, and started to monitor the cells with time-lapse microscopy captured at 3-minute intervals for 72 hours. The dashed arrows point to cells that underwent endoreduplication, eventually causing mitotic catastrophe. An open arrow points to cell that underwent mitotic catastrophe. D, mitotic cells were scored by their transition to a small rounded morphology, followed by cytokinesis and cell duplication. E, dead cells were counted from undivided DOX− and DOX+ cells. C and D data represent four independent experiments. Data are mean ± SE (n >100).