Fig. 5

MASTL depletion causes mitotic catastrophe. MCF7 cells were transfected with either control siRNA or MASTL.5 siRNA for 48 h. a MASTL-depleted cells were scored for abnormal mitosis (≥ 200 mitotic cells for each data point, n = 4, error bars show ± SD). b Representative images of MASTL-depleted cells stained with DAPI (blue). c The intensities of γ-H2AX were scanned and determined by IN Cell Analyzer HCA System. The intensities of 50 dots in the control and MASTL-depleted cells were determined by the γ-H2AX intensity from 200 ~ 700 cells. d The time of mitotic entry was determined by observing mitotic cell rounding with signs of DNA condensation (red arrow) and was monitored by time-lapse microscopy. e After control (Ctrl) siRNA or MASTL.5 siRNA transfection for 24 h, the cells were quantified in four statuses (normal, mitotic arrest, mitotic catastrophe, and mitotic slippage). f MCF7 and T47D cells were transfected with either control siRNA or MASTL.5 siRNA for the indicated time periods. The cells were analyzed by immunoblotting with the indicated antibodies. g MCF7 cells were transfected with either 5 nmol/l control siRNA or 5 nmol/l MASTL.5 siRNA and then incubated without or with zVAD for 48 h. The cell viability was determined by WST-8 assay. h PP2A-Aα/β proteins were immunoprecipitated using an anti-PP2A-Aα/β antibody and analyzed for PP2A activity. i MCF7 cells were transfected with the indicated siRNAs targeting MASTL.5 or PP2A-Aα/β for 48 h. The cells were analyzed by immunoblotting with the indicated antibodies. The data represent typical results and are presented as the mean ± standard deviation of three independent experiments; Scale bars = 10 μm. **P < 0.01