In vitro permeability assay

To measure the permeability of endothelial cells in vitro, we used two different methods in this study: the transwell permeability assay and real-time cell analysis (RTCA) [68]. For the transwell permeability assay, cells (2 x 10⁵) were grown on a Transwell insert (0.4 μm; Corning B.V. Life Sciences, The Netherlands) until a monolayer was formed. The upper chambers were reconstituted with 10% FBS-containing medium with 293T-NS1, S2-NS1 and the inhibitors. At the indicated time points, the media in the upper chambers were changed to 300 μl of serum-free media containing 4.5 μl streptavidin-horseradish peroxidase (HRP) (R&D Systems, Minneapolis, MN). The medium (20 μl) in the lower chamber was collected 5 min after adding streptavidin-HRP and was assayed for HRP activity by adding 100 μl 3,3',5,5'-tetramethylbenzidine (TMB) substrate (R&D Systems). Color development was detected by a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm.

RTCA was used to test cell-cell or cell-matrix adhesion by detecting the electric resistance of the monolayered endothelial cells. High resistance indicates strong endothelial barrier function. Using this device allowed us to detect the kinetic changes in endothelial permeability. For RTCA, 1 x 10⁴ HMEC-1 cells were grown on a 96-well E-plate to form a confluent monolayer. After 293T-NS1 and the inhibitors were added, the resistance of the monolayer was recorded by an xCELLigence Real-Time Cell Analysis System (Cambridge Bioscience, UK) for 24 h.