Figure 7
AMPK activity is involved in protection against ethanol-induced apoptosis. (a) Flow cytometric analysis of apoptotic cells by annexin V–propidium iodide staining. Primary cultures of fetal (GD 17.5) hippocampal neurons were treated with fresh growth medium without (Control) or with indicated combinations of ethanol (100 mM), osmotin (0.16 μM), adiponectin (0.1 μM), Compound C (20 μM), or metformin (5 mM) supplements for 24 h and then analyzed. Cells positive for either Annexin V (early apoptotic) or both annexin V and propidium iodide (late apoptotic) were counted and they lie in the quadrants indicated by the red oval. Shown in the bottom right corner are the results of immunoblot analysis of phospho-AMPK (P-AMPK) levels in these cells at different times after osmotin (0.16 μM) treatment. P-AMPK signals were normalized to β-actin signals and expressed as mean±S.D. (n=5). Asterisk indicates significant difference from time zero group at P<0.01 (b) Shown are quantitative results of the flow cytometric analysis (n=3), expressed as the percentages of the apoptotic cells in the population. (c) Shown are results of western blot analysis of cell extracts of the hippocampal neurons at the end of these treatments. Cell extracts were fractionated by SDS-PAGE and analyzed for caspase-3 and β-actin by immunoblotting. Caspase-3 signals were normalized to β-actin signals and expressed as mean±S.E. (n=5). Sample pairs that are significantly different from one another at P<0.05 are indicated
