FIGURE 8.
Antiapoptotic BH3 domain protein Bcl-xL abolished the effect of SOUL on H2O2-treated NIH3T3 cells. Bcl-xL was overexpressed by co-transfecting or not (Control) sham-transfected (pcDNA) and SOUL-expressing (SOUL) NIH3T3 cells with Bcl-xL-expressing vector (Bcl-xL). 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 Bcl-xL in the cells was performed by Western blotting utilizing anti-SOUL and anti-Bcl-xL 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 Bcl-xL (white bars), or SOUL plus Bcl-xL (dark gray bars). Survival was measured by the MTT method and was expressed as a 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. Pie charts demonstrate the distribution of living (white), necrotic (gray), and apoptotic (black) cells. Values are means of three independent experiments.