Figure 1.

SOCS3 knockdown augments IL-6 or HKSA-induced endothelial permeability and inflammation. HPAECs transfected with control nonspecific (NS) or SOCS3-specific siRNA (si-SOCS3) were stimulated with vehicle (Veh), IL-6 (40 ng/ml) or HKSA (5 × 10⁸ particles/ml). A, TER was monitored for indicated time periods. B, HPAECs on plates coated with biotinylated gelatin were transfected with NS or si-SOCS3 followed by HKSA treatment, 6 h. XPerT assay was performed as described in Experimental procedures; FITC-avidin bound to gelatin-coated base was visualized by immunofluorescence microscopy. DAPI counterstaining shows cell nuclei. Bar: 50 μm. Inset: imaging analysis of FITC-avidin fluorescence signal; n = 4, ∗p < 0.05. C, western blot (WB) analysis of phospho-NFκB, VCAM-1, and ICAM-1 levels in control and HKSA-stimulated HPAEC. Membrane reprobing with SOCS3 antibody was used to verify siRNA-mediated knockdown; β-tubulin served as a loading control. The bar graph depicts the quantitative densitometry of western blots; n = 5, ∗p < 0.05. D and E, VE-cadherin immunostaining was performed visualize adherens junctions; F-actin was stained using Texas Red phalloidin. Gaps in cell monolayers are marked by arrows. Bar: 10 μm. Bar graphs depict quantitative analysis of intercellular gaps (D); n = 4, 10 microscopic fields per condition; ∗p < 0.05. FIU, fluorescence intensity units; RDU, relative density units.