The effect of DFP on mitochondrial LIP, redox potential, cellular ROS production, MMP depolarization, and apoptosis in frataxin-deficient cells. (A) T-rex cells treated (Tet⁺) or not with tetracycline for 6 days were labeled with DHR and, where indicated, supplemented with 50 μM DFP. Mitochondrial LIP was quantified (n = 3, percentage change) using H₂O₂ as in Figure 1. H₂O₂ produced a significant (P < .05, 1-tailed paired t test) increase in DHR fluorescence only in Tet⁺ cells untreated with DFP. (B) Mitochondrial redox potential was monitored by fluorescence microscopy and quantified as in Figure 2A. Mitochondria were significantly more oxidized in Tet⁺ cells untreated with DFP (P < .05, 1-way ANOVA with Tukey post-hoc test). (C) T-rex cells cultured (or not) with Tet for 6 days were treated (or not) overnight with 50 μM DFP and colabeled with 10 μM CDCF-DA–AM (ROS generation) and 50 nM TMRE (mitochondrial potential, MMP) followed by flow cytometric analysis. The medians of CDCF and TMRE fluorescence intensities were significantly different (P < .05) between Tet⁺ and untreated cells. Medians of TMRE fluorescence were not significantly different between untreated and Tet⁺ cells treated with DFP. Medians of CDCF fluorescence were significantly lower (n = 3, P < .05, paired 2-tailed t tests) in Tet⁺ cells treated with DFP compared with untreated cells. (D,E) Untreated and 6-day culture cells were left untreated (D) or exposed overnight to 0.3 μM staurosporine (E) in the presence or absence of 50 μM DFP (also overnight). After staining with annexin V–PE–Cy5, the cells were subjected to flow cytometric analysis to determine the percentage of annexin V⁺ (apoptotic) cells. Only Tet⁺ cells had significantly higher apoptotic indices (n = 3, P < .05, 1-way ANOVA with Tukey post-hoc test).