Fig. 1

Sac-1004 blocks IL-1β-induced BBB disruption in HBMECs. HBMECs were starved and treated with or without Sac-1004 (10 μg/ml, 1 h) prior to stimulation with IL-1β (10 ng/ml, 3 h). Sac-1004 blocked both the TEER decline (a) and the increase in FITC-dextran transendothelial permeability (b) induced by IL-1β. TEER was measured using Millicell ERS-2 (Millipore). For the permeability assay, FITC-dextran was added to the upper chamber. Absorbance of the solution in the lower chamber was measured at 492 nm (excitation) and 520 nm (emission) in a FLUOstar Omega microplate reader. HBMECs were starved and treated with or without Sac-1004 (10 μg/ml, 1 h) prior to stimulation with IL-1β (10 ng/ml, 2 h) (c). Cells were then fixed, permeabilized, and subsequently immunostained for ZO-1, occludin, claudin-5, and F-actin. Rectangle: the region enlarged in high-power images. Translocation of tight junction proteins was assessed as described in the methods section (d). Whole-cell lysates, Triton X-100-insoluble and soluble fractions were subjected to SDS-PAGE followed by western blot analysis with anti-ZO-1, anti-occludin, anti-claudin-5 and anti-actin. Blots are representative of three independent experiments. All data are presented as means ± SEM. ***P < 0.001