Effects of soluble forms of recombinant tumor necrosis factor-related apoptosis-inducing ligand:iron superoxide dismutase (sTRAIL:FeSOD) on mitochondrial membrane potential (ΔΨm). (A) and (B) ΔΨm was measured with (A) rhodamine123 (Rh123) and (B) JC-1. After treatment with sTRAIL:FeSOD (1,000 ng/ml), sTRAIL:mFeSOD (1,000 ng/ml) or FeSOD (500 ng/ml) for 6 hours, cells were incubated for 30 minutes at 37°C with 10 μM Rh123 or 1 μg/ml JC-1 in phosphate-buffered saline and then subjected to flow cytometric analysis. For each sample, 10,000 events were acquired. Results are expressed as a ratio of the relative fluorescence intensity. Each bar represents the mean ± SE obtained from three independent experiments (*P < 0.05 vs. untreated control). (C) Kinetic relationship of redox state (indicated as the glutathione (GSH)/reactive oxygen species (ROS) ratio) and ΔΨm after treatment with sTRAIL:FeSOD. Human promyelocytic leukemia (HL-60) cells or erythroleukemia (K562) cells were treated with sTRAIL:FeSOD (1,000 ng/ml) for 0, 1, 2, 3, 4, 5 or 6 hours and then subjected to ROS, GSH and ΔΨm (JC-1) analysis as described above. (D) Analysis of mitochondrial cytochrome c release. Cells were also incubated for 6 hours with or without sTRAIL:FeSOD (1,000 ng/ml). Cytosolic (Cyto) and mitochondrial (Mito) fractions were prepared from these cells and assessed by Western blot analysis.