Collagen type 1

For immunostaining, the following antibodies were used: Oct-4 (Santa Cruz; sc-5279), proSP-C (Sigma-Aldrich; AB3786), AQP5 (Sigma-Aldrich; AB15858), E-cadherin (BD Bioscience; 610182), collagen type I (Sigma-Aldrich; AB765P), phycoerythrin (PE)-conjugated anti-CD157 (BioLegend, 140203), FITC-conjugated anti-CD54 (BD Bioscience, 561898), and allophycocyanin (APC)-conjugated anti-CD45 (BioLegend, 103111). For Western blot and immunoprecipitations, the following antibodies were used: SPARC (Cell Signaling Technology; #5420, #8725), collagen type I (Sigma-Aldrich; AB765P; Santa Cruz, sc-59772), and a β-actin (Sigma-Aldrich; A5441). Secondary antibodies conjugated to HRP were purchased from Jackson ImmunoResearch. Secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647 were purchased from Life Technologies.

An appropriate volume of collagen type I coating buffer containing 50 µg/ml collagen type I (Sigma-Aldrich, Merck) dissolved in 0.02 M acetic acid was added to cover the growth area of culture vessels and then incubated for at least 1 h at 37°C and 5% CO₂. The coating buffer was aspirated and the culture vessels rinsed three times with PBS prior to their use for BOEC generation and maintenance.

Collagen type I was obtained from Sigma-Aldrich and dissolved in 0.1 M acetic acid to get a 0.1% collagen solution, stored at -20 °C.
The plates were covered with Collagen type I to induce cells’ adherence to the wells’ surface. Collagen solution was diluted with water to a final concentration of 0.01% and applied to wells. The plates were incubated overnight at 4–8 °C. After this, wells were washed with PBS three times, and leaves were stored at 4–8 °C for one month. Before use, the plates were irradiated with UV for 30 min.

To assess adhesion of mouse BMDMs, 96-well plates coated with 2.5 μg type 1 collagen (from rat tail, Sigma) per well and blocked with PBS containing 0.5% BSA were used. Assays were performed by plating 5 × 10⁴ CFDA-SE-labeled (Molecular Probes) BMDMs per well. After 30 min incubation, non-adherent cells were removed by washing with PBS and fluorescence emitted by adhering cells was measured (excitation: 492 nm; emission 517 nm) using a SpectraMax Paradigm Multi-Mode Microplate Detection reader (Molecular Devices).

Cells were seeded at 20,000/well in 24-well semi-permeable plates (Zell Kontakt; Nörten-Hardenberg, Germany) pre-coated with type 1 collagen (Sigma Aldrich, St. Louis, MI, USA) and allowed to reach 100% confluence. On day 2 after seeding, wound healing was initiated by scratching the wells from top to bottom with a sterile 100 μL cone (TipOne; Starlab, Orsay, France). Detached cells were removed by changing the culture medium. The cells were then incubated with a medium (control), 5 µM macitentan, 5 µg/mL pazopanib or 1 µM acriflavine and exposed to N, IH or SH for 7 days. Wound repair was assessed for 7 consecutive days using a camera-coupled optical microscope (×10 magnification Olympus CK2; Olympus, Center Valley, PA, USA). Repaired areas were determined by subtracting any cell-free areas at various endpoints from the initial cell-free areas (Image J software) [55 (link)]. The values of the filled areas were normalized to control area values.
On day 7, the culture media and the cells were separately collected and stored at −80 °C for biochemical analysis and transcript quantification, respectively.