Effects of various chelators on metabolic parameters and mitochondrial DNA content in frataxin-deficient cells. (A) Mitochondrial LIP was measured as in Figure 2 in T-rex cells induced with 1 μg/mL Tet for 6 days. The chelators DFR or SIH (50 μM in all experiments shown in this figure) were added overnight (and maintained during the experiment); 50 μM H₂O₂ supplementation led to a significant increase in fluorescence only in untreated Tet⁺ cells (n = 3, P < .05, one-tailed paired t test). (A) Cells treated overnight with 50 μM of the chelators indicated and induced with 1 μg/mL tetracycline for 6 days were harvested, detergent-extracted, and subjected to luciferase-based ATP assay. ATP content was significantly lower only in Tet⁺ cells without or with SIH treatments (n = 3, P < .05, 1-way ANOVA with Dunnett post-hoc test). (B) Untreated and Tet⁺ (6-day culture) cells were treated overnight with 50 μM of the chelators indicated. Their total DNA was extracted and subjected to real-time quantitative PCR with primer pairs for the mitochondrial DNA markers cytochrome b and the control gene β-actin. A significant decrease in cytochrome b DNA content was observed only in Tet⁺ cells alone and Tet⁺ cells treated with SIH or DFR (P < .05, one-way ANOVA with Dunnett post-hoc test). (C) Cells treated as indicated (t+, 6-day Tet, chelator treatments are 50 μM overnight) were resuspended in full medium, and their oxygen consumption was recorded online with a Clark electrode; CCCP, 50 μM CCCP was added at the indicated time. The oxygen levels in the reaction vessel are given as values normalized to those attained at time 0 (after normalization to milligrams of protein). The calculated rates of oxygen consumption are indicated in panel D. Significant decrease in oxygen consumption was only observed in Tet⁺ cells without and with DFR and SIH (n = 3, P < .05, 1-way ANOVA with Dunnett post-hoc test).