Figure 1.

EtOH reprograms FOXO3 selectively enhancing pro-apoptotic effects. (a) Huh7.5 cells were cultured with or without 50 mM EtOH for 48 h. ChIP assay was performed with anti-FOXO3 antibody. Promoters assessed were p19, p27, SOD2 (superoxide dismutase 2), PrxIII (peroxiredoxin III), Bim (Bcl-2-interacting mediator of cell death), TRAIL (TNF-related apoptosis-inducing ligand), FOXO3, FOXO1 and FOXO4. (b) Huh7.5 cells were transfected with HA-tagged FOXO3, treated with or without 50 mM EtOH for additional 48 h, and ChIP assay was performed with anti-HA antibody. (c and d) mRNA (c) and protein (d) measurements of FOXO3 target gene expression were evaluated by real-time RT-PCR and western blot, respectively. (e) Cell death was evaluated by TUNEL assay in cells treated with 50 mM EtOH either with or without FOXO3 transfection. (f and g) Effects of TRAIL receptor antagonists on FOXO3/EtOH-induced LDH release and caspase activity. FOXO3-transfected cells were treated with or without EtOH in the presence of TRAIL receptor antagonists DR4 (1 μg/ml) or DR5 (1 μg/ml) or the combination (DR4+5). Values are mean±S.D. of three independent experiments. *P<0.05, **P<0.01, ***P<0.001, Student's t-test