Role of the PKC-α/P115RhoGEF/RhoA pathway in TNF-α-induced F-actin rearrangement and BMEC barrier dysfunction. (A) F-actin remodeling and paracellular gap formation were detected by immunofluorescence. Serum-starved cells were transfected with vector-1, n19RhoA, vector-2, P115-shRNA and PKCa-shRNA for 3 h with either DMEM (left) or TNF-α (right). Before stimulation, Bend.3 cells did not display stress fibers although they exhibited an extensive cortical actin network (Figure 5A.a,e). After 3 h of TNF-α exposure, cells exhibited prominent stress fiber formation and paracellular gaps (Figure 5A.b,f), which were partially prevented by inhibition of RhoA activation (Figure 5c-d), and depletion of p115RhoGEF (Figure 5g-h) as well as PKC-α expression (Figure 5 i-j). (n = 3 independent experiments). (B) Vector-2, P115-shRNA and PKCa-shRNA transfected cells were treated with or without TNF-α for various time periods. Bend.3 monolayer permeability was determined by TER. The results show that knockdown of p115RhoGEF and PKC-α can alleviate TNF-α-induced barrier breakdown. *: p < 0.05 vs. vector-2, △: p < 0.05 vs. vector-2 +TNF-a, n = 4/group.