Fig. 6: Acetylmimetic K39Q Cytc expressing cells show increased respiration, mitochondrial membrane potential, and ROS production under basal conditions and increased mitochondrial membrane potential but not ROS production after oxygen-glucose deprivation/reoxygenation.
A Representative western blot of Cytc double knockout cells stably transfected with EV, WT, K39R, K39Q, and K39E constructs immunoprobed for Cytc and loading control GAPDH (n = 3). B ATP levels in the stable cell lines (n = 3). C Mitochondrial stress test measured in Seahorse media supplemented with 10 mM glucose and 10 mM pyruvate was performed via sequential injections of 1 µM oligomycin, 2.5 µM carbonylcyanide-3-chlorophenylhydrazone (CCCP), and 1 µM rotenone/antimycin A (n = 4). D Basal oxygen consumption rate (OCR) from mitochondrial stress test (n = 4). E Mitochondrial membrane potentials (ΔΨm) of cells stably expressing WT, K39R, K39Q, K39E Cytc, and EV were measured using the red/green fluorescence ratio of the JC-10 probe (n = 9). F Mitochondrial ROS production of cells stably expressing WT, K39R, K39Q, K39E Cytc, and EV were measured using MitoSOX fluorescence and normalized to total protein content (n = 9). G, H ΔΨm and mitochondrial ROS production were measured as above at normoxia (gray bars) or after 90 min of oxygen-glucose deprivation followed by 30 min of reoxygenation (red bars) (n = 9). Data are represented as means ± standard deviation. A one-way ANOVA comparing the mean of each mutant with the mean of the control mutant (WT) with the Dunnett post-hoc test was used. For G and H, a student’s two-tailed t-test assuming equal variance was used to compare the normoxia vs. OGD/R condition within each mutant.
