Increase of total uptake of ⁵⁹Fe and localization of ⁵⁹Fe into mitochondria by MtFt overexpression. (A) Total iron uptake. Clone B cells (30% confluence), grown for 24, 48, or 72 hours without (□) or with tetracycline (), were incubated for 24 hours with 1 μM ⁵⁹Fe-Tf and washed 3 times in PBS, and their radioactivity was measured. (B) Distribution of iron between cytosol and mitochondria. The cells treated as described in panel A were homogenized in lysis buffer (sucrose 0.25 M, 10 mM HEPES, pH 7.4, 0.15% bovine serum albumin). The cytosolic and mitochondrial fractions were prepared by differential centrifugation and their radioactivities were measured. The amount of iron in mitochondria is expressed as a percentage of total ⁵⁹Fe taken up. ^*P < .01 compared with 48-hour tet-on cells; ^**P < .01 compared with 72 hour tet-on cells. Data shown are the means ± SD of triplicate determinations from a typical experiment that was performed 3 times.