Fig. 3.

Effects of Rho inhibition on IL-6-induced EC barrier compromise. Human pulmonary artery endothelial cells (HPAEC) were pretreated with vehicle or Y-27632 (2 μM, 30 min) followed by stimulation with IL-6/SR (40 ng/ml/10 ng/ml). A: phosphorylation of myosin-binding subunit of myosin-associated phosphatase type 1 (MYPT1) and myosin light chain (MLC) was detected by Western blot with corresponding phospho-specific antibodies. Thrombin treatment was used as positive control for Rho activation. Equal protein loading was confirmed by determination of β-tubulin content in total cell lysates. B: TER was measured over the 15-h period. In control experiments cells were stimulated with thrombin (0.3 U/ml) with or without Y-27632 pretreatment. C: actin cytoskeleton remodeling was examined by immunofluorescence staining with Texas red-conjugated phalloidin. Y-27632 pretreatment was 2 μM, 30 min; IL-6/SR stimulation was with 40 ng/ml/10 ng/ml for 5 h. Paracellular gaps are marked by arrows. Shown are representative results of 3–5 independent experiments.