Figure 4.

VacA triggers BAX activation and its translocation to mitochondria. (a) Representative confocal images of wt MEF cells incubated for 24 h with H. pylori CS, either wt or P9A mutant. Cells were immunostained with a rabbit anti-VacA antibody and a secondary FITC anti-rabbit antibody. BAX was detected with a murine monoclonal primary antibody specific for its activated form (clone 6A7) and a secondary TRITC anti-mouse antibody. The images are representative of 60 cells examined from five separate experiments. Scale bars: 10 μm. Mander’s coefficient for VacA and activated BAX colocalization was 0.21±0.0231 in wt H. pylori CS-intoxicated cells versus 0.09±0.00198 in P9A mutant CS-treated ones (n=5 independent experiments, 60 cells per experiment). (b) Mitochondria were purified from wt MEF cells incubated with wt or P9A mutant H. pylori supernatants for the indicated time points; 15 μg of mitochondrial proteins were separated by SDS-PAGE and immunoblotted; BAX was revealed with a rabbit anti-BAX antibody. An anti-COX II monoclonal antibody was used as control for equal loading. Cells not exposed to H. pylori CS are defined as untreated cells