Arecoline decreased membrane expression of ZO-1 in confluent TM4 cells. These were treated with indicated arecoline concentration for 6 hours. (A) Morphology of confluent TM4 treated with (right panel) or without (left panel) 400 μM of arecoline were visualized. Treated cells (right panel) displayed flat and spread morphology compared to controls (left panel). (B) ZO-1 protein expression detected by immunofluorescent assay was visualized by fluorescent microscope. Arecoline-treated cells were manifested lower ZO-1 signal than controls. (C) Insoluble and soluble fractions of cell lysates were prepared for Z0-1 detection by Western blot. (D) Inhibitory effect of arecoline on insoluble ZO-1 is plotted on bar graph. Signals of insoluble ZO-1 were quantified by densitometry analysis and expressed as average percentage of respective control cells from three independent experiments to rate arecoline’s inhibitory effect (*P < 0.05 versus 0 μM controls). (E) The amounts of ZO-1 mRNA were determined using RT-qPCR. Results depict mean ± SD of three independent experiments. *P < 0.05 compared with cells incubated without arecoline treatment (0 μM).