Figure 3.

Effect of siramesine on mitochondria. (a) Effect of siramesine on the transmembrane potential of mitochondria. HaCaT and U-87MG cells were treated with different siramesine concentrations for the indicated periods of time. Pancaspase inhibitor Z-VAD-FMK (20 μM), cathepsin inhibitor E-64d (10 μM) and the lipophilic antioxidant α-tocopherol (0.3 mM) were applied 2 h before siramesine treatment. After siramesine treatment, all samples were stained at once with JC-1 at a final concentration of 3.5 μg/ml and analysed with a flow cytometer. Red cells were considered cells with intact mitochondria. The experiments were performed in duplicate; the bars represent mean±S.D. (b and c) Effect of siramesine and α-tocopherol on the release of cyt c into the cytosol. After siramesine treatment, the cytosolic lysates were collected at the indicated time points followed by SDS-PAGE, western blotting and immunodetection of cyt c. Z, Z-VAD-FMK; E, E-64d; T, α-tocopherol; S, siramesine; cyt c, cytochrome c