Figure 5. Modulation of Foxo3a and p47hox using SiRNA-SIRT1 transfection under high-glucose conditions.
Cell were transfected using lipofectamine with prevalidated siRNA-SIRT1. Subsequently, human monocytic cells (1 × 105 cells/ml) were cultured in presence of NG and HG conditions in absence or presence of resverarol (3–12 µmol/L) for 48 h. Then, cells were washed with phosphate-buffered saline (PBS) and then harvested. (A) NG, HG and HG-resveratrol treated cells (transfected with siRNA to SIRT1) were incubated with 5 µmol/L dihydroethidium (DHE). Cells (1 × 105) were stained with 5 µmol/L dihydroyethidium for 30 minutes at 37°C and were washed and resuspended in PBS. The oxidative conversion of dehydroxyethidium to ethidium was measured by flow cytometry. (B) For the western blot analysis, protein was subjected to SDS-PAGE and used p47phox antibody (1:1000 dilution) as detailed in Materials and Methods Section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The immunoblot shown here are representative of three independent experiments with similar results.