Monolayer permeability was assessed as described previously49 (link). Briefly, transwell inserts (Corning, Corning, NY) were coated for 1 hour at 37°C with 0.1 mg/mL bovine skin collagen (Sigma), washed with water 2 times and rinsed with medium prior to plating cells. Cells (4×105 in 100 μl complete EGM-2 medium) were plated in the top chamber and 700 μl of medium was added to the bottom chamber 24 hours prior to the addition of fresh medium to both chambers, with or without 10 or 20 ng/mL BMP9 in the upper chamber (as indicated). After 30 minutes, 25 nM horseradish peroxidase was added to the top chamber, with or without 0.4ug/ml LPS (Sigma), 1U/mL thrombin (Sigma) or 50 ng/mL TNFα (R&D Systems). Medium (20μl) was collected from the lower chamber every 30 minutes for 2 hours. HRP content of the media samples was determined by measuring absorbance after the addition of 150μl of o-Phenylenediamine dihydrochloride buffer to each well of a 96-well plate.
For VE-cadherin assays, PAECs were cultured in EBM-2 media (Lonza) with 2% FBS for 24 hours with or without LPS, thrombin, TNFα and/or 10 ng/mL BMP9. Cells were then stained with FITC-conjugated rabbit polyclonal anti-VE-cadherin (cat #: LS-C40121, 1:100, Lifespan Biosciences, Seattle, WA).
For VE-cadherin assays, PAECs were cultured in EBM-2 media (Lonza) with 2% FBS for 24 hours with or without LPS, thrombin, TNFα and/or 10 ng/mL BMP9. Cells were then stained with FITC-conjugated rabbit polyclonal anti-VE-cadherin (cat #: LS-C40121, 1:100, Lifespan Biosciences, Seattle, WA).