Fig. 1.

GSTpi inhibited TNF-α-induced VE-cadherin internalization and monolayer permeability increases in human umbilical vein endothelial cell (HUVEC). HUVECs were transfected with pcDNA3.1 or Flag-GSTpi and further treated with TNF-α (50 ng/mL). (A) Levels of phospho-VE-cadherin was measured and calculated. VE-cadherin internalization was measured by (B) immunofluorescence (scale bar = 25 μm, the yellow arrowheads indicate the internalization of VE-cadherin), (C) analysis of cell membrane and cytoplasmic VE-cadherin protein levels using immunoblot and (D) flow cytometry (TNF-α (50 ng/mL) for 30 min). (E) The cell indexes of HUVECs were recorded and calculated Endothelial cell permeability was measured, (F) endothelial cell permeability and (G) transepithelial electrical resistance (TEER) were measured and calculated (TNF-α (50 ng/mL) for 1 h). *, compared with the pcDNA3.1 group; #, compared with the pcDNA3.1 + TNF-α group). HUVECs were transfected with NC-siRNA or GSTpi-siRNA and treated with TNF-α (50 ng/mL). (H) Levels of phospho-VE-cadherin was measured and calculated. VE-cadherin internalization was measured by (I) immunofluorescence (scale bar = 25 μm, the yellow arrowheads indicate the internalization of VE-cadherin), (J) analysis of cell membrane and cytoplasmic VE-cadherin protein levels by immunoblot and (K) flow cytometry (TNF-α (50 ng/mL) for 30 min). (L) Cell indexes, (M) endothelial cell permeability and (N) TEER were measured and calculated (TNF-α (50 ng/mL) for 1 h). *, compared with the NC-siRNA group; #, compared with the NC-siRNA + TNF-α group). Data are expressed as means ± SDs (n = 3). Data were analyzed by unpaired Student's t-tests. * or #, P < 0.05; ** or ##, P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)