Effect of frataxin deficiency on cellular labile iron pools. (A) T-rex cells induced with 1 μg/mL tetracycline for the indicated number of days were lysed, and 50 μg of cell lysates was subjected to SDS-PAGE and immunoblotting using antiactin (top panel) and antifrataxin (bottom panel) antibodies. (B) Mitochondrial LIP. Fluorescence images of noninduced cells and cells at day 6 after tetracycline induction (Tet⁺). Fluorescence measured after H₂O₂ was significantly higher (P < .05, one-tailed, paired t test) in tetracycline-induced cells. (C) Mitochondrial LIP was measured in DHR-loaded T-rex cells exposed to 50 μM H₂O₂ for 20 minutes, after induction with tetracycline for the indicated number of days (d). Shown are mean fluorescence intensities (as percentage of basal) based on n = 3 experiments. (D) Cytosolic LIP of T-rex cells induced with tetracycline for the indicated number of days was measured by the calcein method. Calcein fluorescence was monitored continuously before and after the addition of SIH, added to reveal the entire cellular pool of calcein-bound iron, termed LIP. Shown is normalized calcein fluorescence, relative to the initial fluorescence, plotted against time for untreated cells 0 days (----) and for cells exposed to Tet for 2 days (□), 4 days (○), 6 days (●), and 9 days (*). The bars on the right denote the fluorescence recovered after SIH addition, which is proportional to cytosolic LIP. Frataxin-deficient (Tet⁺) cells showed significantly lower cytosolic LIP compared with untreated cells (P < .05, 1-way ANOVA with Dunnett post-hoc test).