Fig. 3.

MFF regulation of mitochondrial outer membrane permeability. (a–c) PC3 cells were transfected with vector, Flag-MFF1 (a), Flag-MFF2 (b) or Flag-MFF5 (c), immunoprecipitated (IP) with an antibody to Flag and analysed by Western blotting. Sup, supernatant; Unb, unbound. (d) PC3 cells transfected with siCtrl or siMFF (top) or treated with H₂O₂ (bottom) were labelled with calcein in the presence of CoCl₂ and analysed by flow cytometry. Numbers indicate fluorescence units per each condition. None, untreated. (e and f) PC3 cells transfected with siCtrl or siMFF were incubated with Fluoro3-AM and FLUO-5 N or, alternatively Rhod2-AM, and changes in mitochondrial (Mito)-, endoplasmic reticulum (ER)- or cytosolic (Cytosol)-associated Ca 2+ levels were determined by flow cytometry (e, representative experiment) and quantified (f). Numbers correspond to fluorescence units. Mean ± SD. (g) DU145 (top) or PC3 (bottom) cells were transfected with siCtrl or MFF-directed siRNA (siMFF) and analysed for mitochondrial inner membrane potential by TMRE staining and flow cytometry with or without suboptimal concentrations of the uncoupler, FCCP. Representative experiment (n = 3). (h) The conditions are as in (g) and TMRE staining was quantified in siRNA-transfected DU145 and PC3 cells. Mean ± SD.