Protease inhibitor 3

Cell cultures were lysed in RIPA buffer (Thermo Scientific) containing 0.5% Protease Inhibitor III (Merck) and 1 mM Sodium Orthovanadate (Sigma). Samples were electrophoresed and then transferred to a PVDF membrane. Membranes were probed with primary antibodies overnight at 4 °C, followed by HRP-conjugated secondary antibodies. Antibody information is provided in the Supplemental Material. Protein was visualised using enhanced chemiluminescence reagents (ECL, GE Healthcare), according to manufacturer's instructions and quantification was performed using Image J. For western blot analysis following treatment with type I collagen, tissue culture plastic was coated with type I collagen (Sigma, 3, 6 and 10 mg/ml) and then pre-incubated in MSC basal medium overnight. Cells were seeded at a density of 2.1 × 10⁴ per cm² and incubated for 18 h before analysis by Western Blotting.

Mouse trachea was exposed and lungs were lavaged twice with 2% FCS in PBS using a 20-gauge i.v. catheter (Terumo). Cells were obtained from BALF by centrifugation at 400g for 5 minutes at 4°C. RBCs were lysed with RBC lysis buffer (Omics Bio), and cells were spun onto slides and stained with Diff-Quick solution (Polysciences, Inc.). The BALF cellular profile was assessed by differential cell counting. For ELISA measurement, lavage was performed with PBS supplemented with protease inhibitor III (Merck), phosphatase inhibitor II (Merck), and phosphatase inhibitor III (Merck).