Fig. 3. p31^comet controls the timing of mitotic slippage by regulating MCC.

a Mitotic slippage is delayed after knockdown of p31^comet. HeLa cells expressing BCL-2 were transfected with either control or siRNA against p31^comet (sip31). Knockdown of p31^comet was confirmed with immunoblotting (Fig. S3A). The cells were synchronized and treated with NOC as described in Fig. 1c. Individual cells were then tracked using live-cell imaging. The duration of mitotic arrest and the elapsed time between mitotic entry and mitotic cell death/slippage are shown. The live-cell imaging profile of individual cells is shown in Fig. S3B. b Mitotic slippage is delayed in p31^comet-deficient cells. HeLa (WT) and p31^comet-deficient cells (p31^KO) (both stably expressing BCL-2) were incubated with NOC before analyzed using live-cell imaging. The duration of mitotic arrest and the elapsed time between mitotic entry and mitotic cell death/slippage are shown. The live-cell imaging profile of individual cells is shown in Fig. S4A. c MCC inactivation is delayed in the absent of p31^comet. WT and p31^KO (both stably expressing BCL-2) were synchronized and treated with NOC as described in Fig. 1c. The cells were harvested at the indicated time points. Lysates were prepared and subjected to immunoprecipitation (IP) using antiserum against MAD2 followed by immunoblotting analysis. d Knockout of p31^comet delays MPS1i-mediated mitotic slippage. WT and p31^KO (both stably expressing BCL-2) were synchronized and treated with NOC as described in Fig. 1c. At t = 0 h, the cells were treated with the MPS1 inhibitor AZ 3146. Lysates were prepared at the indicated time and analyzed with immunoblotting.