In vitro transwell assay

For the endothelial permeability assay, HMECs (10⁵cells/well) were seeded on gelatin-coated Transwell inserts of 24-well plates, in 250μL complete with Endothelial Cells Growth Medium MV. They were then incubated for 72 h at 37 °C and with 5% CO2. The cells were incubated in free M200 medium for two hours before stimulation. The cells were then incubated with the different compounds, as indicated in the figure legends. After treatment, the upper chamber medium was replaced by 300μL of M200, containing HRP-coupled streptavidin. The lower chamber medium was collected after 10-min incubation at 37 °C, and every condition was aliquoted in triplicate. The TMB substrate was added for 10 min, and 2 N H₂SO₄ was applied to stop the reaction before acquiring 450 nm absorption in an Elisa reader. Resultant absorption intensity values were normalized over the vehicle control condition. Each experiment was repeated three times.