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. 2004 May 7;5(6):626–631. doi: 10.1038/sj.embor.7400154

Figure 3.

Figure 3

Mitotic delay of cells lacking Sgt1 is caused by the spindle checkpoint. (A) Mad1 is absent from the kinetochores of Sgt1-depleted cells despite evident lack of microtubule attachment. (B) Addition of nocodazole did not cause a return of Mad1 to kinetochores. (C) Also BubR1 was missing from the kinetochores of Sgt1-depleted cells. (D) Parallel silencing of Sgt1 and Mad2 by RNAi as revealed by western blot on total cell lysates 72 and 96 h after siRNAs transfection. Mad1, CENP-I and vinculin are shown as controls. (E) Bivariate FC analysis on the indicated samples. White bars: 4N cells; grey bars: 4N cells that were H3-P positive. Dual ablation of Mad2 and Sgt1 relieves the mitotic block caused by Sgt1 ablation. (F) Cells lacking Sgt1 are unable to maintain a mitotic arrest beyond an average of 11 h, even if nocodazole is added. Black bars: Duration of mitosis in cells filmed by time-lapse microscopy from 48 to 96 h after siRNA transfection. (G) Sgt1-depleted cells cannot mount a full-fledge MSC response in nocodazole. White bars: 4N cells; grey bars: 4N cells that were positive for H3-P. The lower percentage of 4N cells in the total cell population after Sgt1 depletion is explained by a G1 arrest, as documented in SF 2. Note that the scale of this figure is different from that used in (E).