Fig. 5.

MFF inhibition of mitochondrial cell death. (a and b) DU145 (top) or PC3 (bottom) cells transfected with siCtrl or increasing concentrations of siMFF (100–200 nM) were analysed by Western blotting (a) and mitochondrial-dependent cell viability was determined by an MTT assay (b). Mean ± SD (n = 3). ***, p < .0001 (by two-sided unpaired t-test). (c) PC3 cells were transfected with siCtrl or siMFF and analysed for cellular morphology by light contrast microscopy. Arrows, cells with membrane blebbing and chromatin condensation. Scale bars, 50 μm. (d) Two independent clones of DU145 or PC3 cells stably expressing shMFF (MFF #1 and MFF #2) or pLKO were analysed by Western blotting. Cl., cleaved. Bar graph, densitometric quantification of total PARP (t-PARP) bands. RU, relative units. (e) DU145 (left) or PC3 (right) cells were transfected with siCtrl or siMFF and analysed for Annexin V and propidium iodide (PI) staining by multiparametric flow cytometry. The percentage of cells in each quadrant is indicated. (f) PC3 cells stably expressing a mitochondrial Keima-Red reporter were transfected with siCtrl or siMFF and analysed for pH-sensitive changes in fluorescence emission (PE-Texas Red) indicative of mitophagy. The percentage of cells in each quadrant is indicated. Representative experiment. BV605, Brilliant Violet 605 (g and h). WT, VDAC1^−/− or CypD^−/− MEF were transfected with siCtrl or siMFF and analysed for cell viability by Trypan blue exclusion and direct cell counting after 48 h (g) or an MTT assay (h). Mean ± SD. ***, p < .0001; **, p = .001; ns, not significant (by two-sided unpaired t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)