TNF-α activates RhoA, which mediates barrier dysfunction in Bend.3 cells. (A) PcDNA3.1hygro-n19RhoA (the dominant negative mutant of RhoA), and PcDNA3.1hygro vector plasmids (vector-1) were transfected into Bend.3 cells by Lipofectamine 2000. Stably transfected cells were obtained using the Hygromycin B (400 ug/ml) selection method after transfection. Untreated Bend.3 cells served as controls. The inhibitory effect of n19RhoA was confirmed by pull-down assay. (B) Levels of active RhoA in vector-1 cells after TNF-α (10 ng/ml) treatment were detected at various time points by pull-down assay to detect relative amounts of RhoA-GTP. Total RhoA levels served as loading controls. The results show that TNF-α induced a rapid activation of RhoA within 1 min, reaching peak activation within 30 min. (n = 3 independent experiments). (C) Vector-1- and n19RhoA-transfected cells were treated with or without TNF-α for various time periods. TER assay was used to determine Bend.3 monolayer permeability. The results show that TNF-α-induced vector-1 cell barrier breakdown was partially alleviated in n19RhoA cells. *: p < 0.05 vs. Vector-1, #: p < 0.05 vs. Control, △: p < 0.05 vs. Vector-1+TNF-a n = 4/group.