Effect of frataxin deficiency on mitochondrial redox potential, ROS generation, and MMP. (A) T-rex cells were transfected with a construct encoding for the redox potential reporting ratiometric roGFP2 protein, which was fused to a mitochondrial targeting sequence. After 6 days with (Tet⁺) or without tetracycline induction, the cells were analyzed by confocal microscopy (left panel), using ratiometric fluorescence at 405/488 excitation ratios (see lookup table in pseudo-image color in the “Tet⁺” image) and 520 nm emission. Scale bar represents 10 μm. (Right panel) Redox potentials calculated from the roGFP2 405/488 excitation ratios normalized to the range between maximal (10 mM H₂O₂) and minimal (10 mM DTT) oxidation, in combination with the nonlinear regression fit to an in situ calibration curve, f = 0.0509 + 0.8305/(1 + exp(−(x = 249.5422)/6.6161)). Mitochondrial redox potential in Tet⁺ cells was significantly higher than in untreated cells (n = 3 experiments, P < .05, one-way ANOVA with Dunnett post-hoc test). (B,C) T-rex cells, treated (Tet⁺) or not with tetracycline for 6 days (left panels) or for the durations indicated (right panels), were labeled with (B) the ROS-sensitive agent CDCF-DA-AM (10 μM) or (C) the MMP potentiometric probe TMRE (0.2 μM). Cells were examined by confocal microscopy using (B) the 405 excitation/488 emission filter set for CDCF (green) or (C) the 543 excitation/633 emission filter set for TMRE (red). (Right panels) Data are from similar fluorescence microscopy analyses of cells treated with Tet for 0 to 9 days. CDCF fluorescence was higher and TMRE fluorescence was lower in Tet⁺ cells compared with noninduced cells (n = 3 experiments, P < .05, one-way ANOVA with Dunnett post-hoc test). The average number of mitochondria punctae per cell in Tet⁺ cells was 235 plus or minus 90, significantly higher (P < .05, unpaired, 2-tailed t test) than in noninduced cells, where it was 66 plus or minus 54. Scale bars represent 10 μm.