Cellular iron incorporation and oxidative damage. (A) Transfected and control cells were incubated for 3 hours with 2 μCi/mL (0.074 MBq) (⁵⁵Fe) ferric ammonium citrate, and then the iron content was analyzed in the total cell extract (Tot), in the postmitochondrial fraction (PMF), and in the mitochondrial fraction (MF). The data are expressed as picomole ⁵⁵Fe per milligram protein. Mean and SD of 4 experiments. (B) The 3 cellular fractions were loaded on nondenaturing PAGE (10 μg protein per lane), and then exposed to autoradiography to analyze protein-bound iron. A single band was evident, and it corresponded to the cytosolic H/L ferritin. The mobility of the mitochondrial ferritin (MtF) is indicated. (C) The mock-transfected controls and siRNA-transfected cells were incubated for 2 hours with various concentrations of H₂O₂, and then their viability was evaluated by MTT assay. Mean and SD of 3 experiments in octuplicate. The difference between the 2 plots was statistically highly significant (P = .009).