Fig. 4. Silencing Cenp-F activates the spindle checkpoint. HeLa cells were transfected with siRNA duplexes designed to repress both Cenp-F and BubR1 then analysed 48 hours later by immunoblotting, flow cytometry and time-lapse microscopy. (A) Blot showing efficient corepression of Cenp-F and BubR1. Numbers indicate size markers in kDa. (B) Bar graph quantitating the number of MPM-2-positive cells in asynchronous populations as determined by flow cytometric analysis of 10,000 cells. Values represent the mean±s.e.m. of three independent experiments. (C) Histograms plotting the time from nuclear envelope breakdown to anaphase. Number of cells analysed; Control, 33; BubR1 RNAi, 36; Cenp-F RNAi, 53; Dual RNAi, 74.