Effect of ethanol treatment on Sestrin3 (Sesn3) and AMP-activated protein kinase (AMPK) phosphorylation in VL-17A cells. Immunoblot analysis of proteins in VL-17A cells treated with different concentrations of ethanol for 48 h is shown. The immunoblot bands (A) were quantified by densitometry analysis, and the ratio of Sesn3 to β-actin (B) and phosphorylated (p)-AMPK-α to AMPK-α (C) were calculated by setting the value of controls as one. Ethanol treatment inhibited the expression of Sesn3 and AMPK-α phosphorylation in the VL-17A cells in a dose-dependent manner. Results are means ± SD (the blot is a representative of 3 blots from 3 individual experiments). Significant differences among group were determined by one-way ANOVA. *P < 0.05, significant difference vs. control. Accumulation of triglyceride (TG) in VL-17A cells after ethanol treatment is shown. The presence of TG was confirmed by Oil Red O staining (D), as well as TG measurement (E). VL-17A cells were transduced with control vectors [AdGFP (green fluorescent protein) or shGFP], AdSesn3 (F) and shSesn3 (G). H: overexpression of Sesn3 ameliorated TG accumulation in VL-17A cells exposed to ethanol (E). Values are means ± SD. *P < 0.05, significant difference vs. respective vector controls.