Oxidative stress susceptibility of frataxin-deficient cells. (A) Cells cultured with Tet for 0 to 9 days were stained with annexin V–PE–Cy5 and analyzed by flow cytometry to determine the percentage of annexin V⁺ (apoptotic) cells. Tet⁺ cells had significantly higher apoptotic indices as tested by one-way ANOVA with Dunnett post-hoc test (n = 3, P < .05). (B) Cells cultured with Tet for 0 to 9 days were treated with 0.3 μM staurosporine, stained with annexin V–PE–Cy5, and analyzed by flow cytometry to determine the percentage of annexin V⁺ (apoptotic) cells. Tet⁺ cells had significantly higher apoptotic indices, as tested by one-way ANOVA with Dunnett post-hoc test (n = 3, P < .05). (C) Glucose oxidase at increasing concentrations (to produce increasing levels of H₂O₂) was added for 24 hours to T-rex cells treated (----) or not (—) with tetracycline for 6 days. Cell viability was assessed after 24 hours by retention of calcein (loaded as calcein-AM). (D) T-rex cells were treated or not for 2, 4, and 6 days with Tet, and IC₅₀ values were calculated (using similar survivorship curves as shown on left) by nonlinear regression. Untreated and Tet⁺ cultures were matched for cell density to minimize the influence of cell density on cellular resistance to glucose oxidase.