Activation of ERK1/2 MAPKs is important for TNF-alpha induction and ZO-1 protein redistribution by arecoline in TM4 cells. (A) PD98059 pretreatment inhibited ERK1/2 activation. Cells were stimulated with or without 400 μM of arecoline for 10 minutes after pretreatment with 10 μM PD98059 for 1 hour, levels of ERK1/2 and phospho-ERK1/2 determined by Western blot. Results shown represent three independent experiments. (B) (C) PD98059 pretreatment inhibited arecoline-induced increase in mRNA (B) and soluble protein (C) of TNF-alpha. Cells were treated with or without arecoline for 6 hours after PD98059 pretreatment. Levels of mRNA and soluble protein of TNF-alpha were measured by RT-qPCR and ELISA respectively. Results depict mean ± SD of three independent experiments. *P < 0.05 compared with cells incubated without arecoline treatment (mock control). (D) PD98059 pretreatment rescued arecoline-induced protein redistribution of ZO-1. Levels of insoluble or soluble ZO-1 were determinate by Western blot. Results represent three independent experiments. (E) Effect of arecoline and PD98059 on insoluble ZO-1 is plotted on bar graph. Signals were quantified by densitometry analysis and expressed as average percentage of respective control cells from three independent experiments (*P < 0.05 versus mock controls).