Am966

Human lung microvascular endothelial cells (HLMVECs, Lonza) were cultured at 37°C in an atmosphere of 5% CO₂ with EGM-2 medium (Lonza) containing 25 mL FBS (5%), 0.5 mL hEGF, 2.0 mL hFGF-β, 0.5 mL VEGF, 0.5 mL ascorbic acid, 0.2 mL hydrocortisone, 0.5 mL R3-IGF-1, and 0.5 mL gentamycin. Phospho (T18/S19)-MLC, MLC, antibodies, and cell lysis buffer were obtained from Cell Signaling. Phospho (Y658)-VE-cadherin antibody was purchased from Invitrogen. VE-cadherin antibody was from Santa Crus Biotechnology. β-Actin antibody, scrambled siRNA, LPA1 siRNA, and LPA were from Sigma Aldrich. LPA1 antibody was obtained from Proteintech. AM966 was from Apex Bio. Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibodies, ECL kit, and SDS-PAGE for western blotting were purchased from Bio-Rad Laboratories, Inc. For immunostaining, anti-mouse Alexa-488, anti-rabbit Alexa-568, and DAPI were from Invitrogen. Transfection reagent FuGENE HD was from Promega. All other reagents were of analytical grade.

All cell culture reagents and consumables were obtained from Gibco (Carlsbad, CA, USA) or Corning Incorporated (Corning, NY, USA). hADMSCs (from a single donor) were purchased from Cyagen Biosciences (Guangzhou, China). All immunophenotypes of the hADMSCs were validated by the manufacturer. d-galactosamine (Gal), lipopolysaccharide (LPS), methylthiazolyldiphenyl tetrazolium bromide (MTT), pertussis toxin (PTX), W146, JTE013, salirasib, and MK-2206 were purchased from Sigma-Aldrich (St. Louis, MO, USA). AM966 and CAY10444 were obtained from APExBIO (Houston, TX, USA) and Cayman Chemical (Ann Arbor, MI, USA), respectively. The NF-κB p65 acetylation inhibitor anacardic acid (AnaAcid) was a product of Abcam (Cambridge, UK). All antibodies and UO126/wortmannin were purchased from Cell Signalling (Danvers, MA, USA).