Arecoline activates ERK1/2 MAPKs and increases TNF-alpha production. (A) Protein signal of phospho-ERK1/2, ERK1/2, phospho-JNK, phospho-IkappaB-alpha, phospho-PP2A, phospho-STAT1 and GAPDH in arecoline (400 μM) treated cells were detected by Western blot. Arecoline induces phosphorylation of ERK1/2 MAPKs. These same membranes were stripped and re-detected by antibodies of total ERK1/2 and GAPDH. Results shown represent three independent experiments. (B) (C) No obvious cell death in arecoline-treated TM4 cells treated with indicated arecoline concentration, cytotoxicity or viability gauged by released LDH activity detection kit after 6 hours (B) or MTS Assay after 6, 12 and 24 hours of treatment (C). Data express percentage of control cells (*P < 0.05 versus 0 μM control group). (D) (E) Arecoline induced TNF-alpha mRNA (D) and soluble protein production (E). Cells were treated with indicated concentration of arecoline for 6 hours, levels of TNF-alpha mRNA measured by RT-qPCR and soluble protein in culture medium measured by ELISA. Results depict mean ± SD of three independent experiments. *P < 0.05 compared with cells incubated without arecoline treatment (0 μM).