FIGURE 6.

Suppression of cyclophilin D abolished the effect of SOUL on H₂O₂-treated NIH3T3 cells. Suppression of CycD was achieved by co-transfecting or not (Control) sham-transfected (pcDNA) and SOUL-expressing (SOUL) NIH3T3 cells with 30 nm specific siRNA-expressing vector (CycD siRNA) according to the manufacturer's recommendation. The cells were treated for 24 h with hydrogen peroxide at concentrations ranging from 0 to 500 μm. A, demonstration of expression of SOUL and cyclophilin D in the cells was performed by Western blotting utilizing anti-SOUL and anti-cyclophilin D as well as anti-actin (loading control) primary antibodies. Photomicrographs demonstrate representative blots of three independent experiments. B, comparison of the effect of H₂O₂ on survival of NIH3T3 cells transfected with pcDNA3.1 (black bars), SOUL (light gray bars), pcDNA3.1 plus CycD siRNA (white bars), or SOUL plus CycD siRNA (dark gray bars). Survival was measured by the MTT method and was expressed as percentage of the survival of untreated double sham-transfected cells. Values are means ± S.E. of three independent experiments. Significant differences are indicated above the bar. *, p < 0.01; **, p < 0.001. C, detection of necrosis and apoptosis was by flow cytometry following fluorescein-conjugated annexin V and PI double staining performed at the end of a 24-h incubation in the presence or absence of 300 μm H₂O₂. D, pie charts demonstrate the distribution of living (white), necrotic (gray), and apoptotic (black) cells. Values are means of three independent experiments.