Fig. 8

Effect of UFH on nocodazole-induced MTs disassembly and actin cytoskeleton remodeling. HPMECs were pretreated with vehicle or UFH followed by nocodazole (0.2 μM) stimulation for 6 h. a MTs remodeling was examined by immunofluorescence staining with Alexa Fluor® 488, (scale bars = 10 μm). b The TEER across the cells were measured. c The influx of FITC-conjugated dextran across the cells were measured. d Actin cytoskeletal remodeling was examined by immunofluorescence staining with TRITC-phalloidin. Arrows indicate intercellular gaps. Vehicle group displayed intact monolayer and stress fibers were rarely observed. UFH pretreatment decreased nocodazole -induced the formation of stress fibers and intercellular gaps, (scale bars = 50 μm). The area of inter-endothelial gaps in the microscope fields was assessed and normalized to the area of gaps induced with nocodazole alone. For each group, total of nine microscope fields from three parallel experiments were analyzed. e Western blot analysis was used to evaluate the levels of Acetylated tubulin, GEF-H1 and phosphorylation of MYPT1. Equal protein loading was confirmed by GAPDH antibody. Data are representative of 3 independent experiments. t-test, * P < 0.05 compared to the vehicle group. t-test, # P < 0.05 compared to the nocodazole -treated group