Fig. 5.

Mitochondrial fragmentation-induced depletion of both hFis1 and Opa1 was capable of restoring the impaired mitotic entry. HeLa cells were transfected with pREP4 construct containing short hairpin RNA (shRNA) of the target sequence of hFis1, Opa1, or both of them together along with control, and the transfectants were selected and synchronized using the double thymidine block (DTB) method. a The elongated mitochondrial morphology in hFis1-depleted cells was reversed by knockdown of Opa1. b Cells were stained with aceto-orcein and the mitotic index was determined. At least 200 cells were counted in each condition and repeated three times to calculate mean standard deviation. c In hFis1-depleted cells, Opa1 levels were determined by Western blotting. d Cells were harvested at each time point after DTB release and the expression levels of cyclin B1 and p-H3 were analyzed by immunoblotting. The graphs represent quantification of phospho-histone H3 (p-H3) expression levels by normalization to actin. e Cell lysates were prepared at the indicated time point after DTB release for cyclin B1/cyclin-dependent kinase1 (Cdk1) kinase activity. Anti-cyclin B1 antibody was used to immunoprecipitate kinase complexes, and histone H1 was used as a substrate. Phosphorylated substrate was detected by autoradiography. *p < 0.05, **p < 0.01 vs. control shRNA by Student’s t test