COE modifies the endogenous level of HSP27 and inhibits TNF-α-induced activation of NF-κB/Snail in SGC-7901 cells. (A) COE suppresses TGF-β1 induced HSP27 expression. SGC-7901 cells were treated with TGF-β1 (10 ng/mL) or with TGF-β1 and COE (5, 10, 20 μg/mL) for 24 h. The expression levels of HSP27 was analyzed by western blot assay. (B) The level of HSP27mRNA was examined by qRT-PCR using total mRNA of SGC-7901 cells and compared with TGF-β1 alone. The data was analyzed using the 2-ΔΔCt method. (C) Cells were treated with COE and/or TGF-β1 (10 ng/mL) in the presence or absence of the proteasome inhibitor MG132 (1 μM). HSP27 content was evaluated by western blot. (D) Effect of COE on TNF-α-mediated activation of NF-κB/Snail signal pathway. Cells were pretreated with COE (5, 10, 20 μg/mL) for 1 h, and then TNF-α (10 ng/mL) was added for another 6 or 12 h. Nuclear and cytoplasmic fractions were separated by a nuclear extract kit, and the expression levels of the NF-κB p65, Snail, IκBα and phosphorylation of IκBα protein were measured by western blot analysis. (E) Effect of COE on TNF-α-induced activation of NF-κB. SGC-7901 cells were transiently co-transfected with pGL6-NF-κB-TA-luc and pRL-TK. After 6 h, the cells were incubated with COE for 1 h followed by TNF-α (10 ng/mL) activation for 24 h. The transcriptional activity of NF-κB was determined by luciferase reporter gene assay and normalized to the internal control. The results are representative of at least three independent experiments. #P <0.05, ##P <0.01, compared with control. *P <0.05, **P <0.01, compared to TGF-β1 or TNF-α-treated cells.