Figure 5. Mortalin regulates thrombin-induced endothelial permeability.

(A) HPAEC transfected with control-siRNA or mortalin-siRNA were seeded at 20,000 cells per transwell insert and cultured for 48 hours. Following this, the confluent monolayer was treated with thrombin (5 U/ml) for 30 minutes. FITC-Dextran permeability testing was done to check monolayer integrity. Permeation was stopped by removing the inserts from the wells. Media from the receiver tray was transferred to a 96 well opaque plate to measure fluorescence. Fluorescent intensities were quantified using a fluorescent plate reader with filters appropriate for 485 nm and 535 nm excitation and emission. The data are the means ± S.E. (n = 4–6 for each condition). (B) Following permeability testing the endothelial monolayers were stained for bright field imaging. (C) HPAEC were treated with 1µg/ml MKT-077 (1 h) before or 1 h after thrombin treatment (5 U/ml; 1 h). In vitro FITC dextran permeability assay was performed to monitor thrombin-induced endothelial permeability.