ABCB7 silencing in MtF-expressing cells. (A) The HeLa cell clone harboring a vector for the expression of MtF (MtF clone) was transfected with ABCB7-siRNA and then incubated with 2 μCi/mL (0.074 MBq) (⁵⁵Fe) ferric ammonium citrate. After 3 hours, the cells were harvested and iron content was analyzed in the total and mitochondrial fractions. Iron incorporation in the mitochondria is expressed as percentage of total incorporated iron. In parallel are shown the data of MtF-free HeLa cells (HeLa) from Figure 5. Data are shown as mean and SD of 3 experiments. (B) The 3 cellular fractions were analyzed on nondenaturing PAGE and exposed to autoradiography to evaluate iron incorporation into cytosolic (H/L) and mitochondrial (MtF) ferritins. (C) Blotting with anti-MtF antibody of the total cellular extracts (30 μg per lane) of control and transfected MtF clone, which recognizes MtF and H-ferritin (HF). Gel densitometry showed that MtF in the ABCB7-silenced cells is 80% of the control cells. (D) In the upper panel, the total cell homogenates (10 μg) were analyzed for SOD activity by nondenaturing PAGE and nitroblue tetrazolium staining. SOD2 and SOD1 indicate activity of mitochondrial Mn SOD2 and of cytosolic SOD1 enzymes, respectively. CB indicates Coomassie blue stain of a band used for calibration of protein load. Lower panel shows SDS-PAGE Western blotting of the total homogenates (10 μg) stained with anti-SOD2 (SOD2) and with antiactin antibody. Data are representative of 3 independent experiments with equivalent results.