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Fig. 3. Overexpressing UbcH10 does not overcome the spindle-assembly checkpoint. (A) HeLa cells that overexpress UbcH10 arrest in mitosis in response to nocodazole at a frequency similar to that of control cells. Cells were treated with 50 ng/ml nocodazole and entry into and exit from mitosis was monitored by time-lapse DIC microscopy with images taken every 15 minutes (not shown). The graph shows the mean values and ±s.e.m. of mitotic cells counted at hourly intervals from three independent experiments are shown. (B) A minority of cells that overexpress UbcH10 undergo mitotic slippage. HeLa cells that had been transfected to express UbcH10 and GFP under an IRES were incubated in 50 ng/ml nocodazole and monitored by fluorescence and DIC microscopy. Images were taken at 5-minute intervals. The cell shown is representative of 24 cells out of 224, and exits mitosis after more than 3 hours. Bar, 10 µm. (C) Cells with reduced levels of Mad2 cannot arrest in mitosis in response to nocodazole. HeLa cells were transfected with 200 nM siRNA targeting Mad2 and 48 hours later monitored by time-lapse DIC microscopy in the presence of 50 ng/ml nocodazole. The cell shown is representative of 56 cells out of 564 cells analysed. Bar, 10 µm.
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