Fig. 5. MARCH5 regulates mitotic apoptosis through BAK.

A Disruption of MARCH5 elevates BAK expression. WT and MARCH5^KO cells were synchronized and arrested in mitosis as before. The expression of the indicated proteins was analyzed with immunoblotting. Note that several proteins (MCL1, BCL2, and BCL-XL) displayed mitotic gel mobility shifts. B Increased expression of BAK during mitotic arrest in MARCH5^KO. WT or MARCH^KO cells were synchronized and arrested in mitosis as before. The expression of BAK during G₂ and mitosis was quantified from immunoblots of three independent experiments (mean ± SEM). C PARP1 cleavage in MARCH^KO-mediated mitotic apoptosis is reduced in the absence of BAK. BAX or BAK was ablated with CRISPR-Cas9 in MARCH5^KO cells. The cells were synchronized and arrested in mitosis as before. Protein expression was analyzed with immunoblotting. D MARCH^KO-mediated mitotic apoptosis is delayed in the absence of BAK. MARCH5^KO, MARCH5^KOBAX^KO, and MARCH5^KOBAK^KO (all were MCL1^KO cells expressing ^mAIDMCL1) were transiently transfected with histone H2B-GFP before synchronized and arrested in mitosis as before. Individual cells were then tracked using live-cell imaging. The duration of mitotic arrest is plotted using Kaplan–Meier estimator. Box-and-whisker plots show the elapsed time between mitotic entry and mitotic apoptosis/slippage. *p < 0.05; ****p < 0.0001.