Mitochondrial Membrane Potential Quantification

Mitochondrial membrane potential (MMP) assays were performed as previously described [15 (link)]. Briefly, yeast cells were cultured and treated with Mel56 as described in the viability assays section. Following Mel56 treatment, the cells were washed with 1X PBS and incubated with the dye JC-10 (Sigma, USA) (final concentration of 10 µM) for one hour at 37 °C. JC-10 fluorescence was quantified by flow cytometry. The cells were treated with 10 µM of the mitochondrial membrane perturbant carbonyl cyanide 4-(trifluoromethoxy) phenyl hydrazone (FCCP; ABCAM, USA) as a positive control to profile cells with depolarized mitochondria. The experiments were repeated at least three times, and data that are presented are representative of one experiment.