Hlmvec

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.

Human lung microvascular ECs (HLMVECs, Lonza, Houston, TX, USA) were seeded into 6-well plates (300,000 cells/well) with complete growth medium. At 48 h after cell seeding when the cells reached 80% confluency, the cells were changed to basal medium supplemented with 2% FBS for 10 h and then treated with the indicated doses of Rabeprazole. Cells were collected for molecular analyses at 18 h post-treatment.

Human Lung Microvascular ECs (HLMVECs) or human umbilical vein endothelial cells (HUVECs) (Lonza, Morristown, NJ) were used in our study.

HPAECs and HLMVECs (Lonza) were grown in EGM-2 medium supplemented with 10% fetal bovine serum (FBS) and EGM-2 MV Bulletkit or EGM-2 Bulletkit (Lonza), respectively. Cells were used between passages 2 and 6. CHO-K1 and HEK293 cells (ATCC) were grown in DMEM with 10% FBS (Gibco). Cells were transfected at ∼80% confluence using X-tremeGENE HP according to the manufacturer’s protocol (Roche) and used after 24–48 hr. For siRNA-mediated inhibition of protein expression, cells were treated with 70 nM siRNA (Supplemental Experimental Procedures) using GeneSilencer transfection reagent according to the manufacturer’s protocol (Genlantis) and used after 72–96 hr. For experiments in which cells were stimulated with α-thrombin, the FBS concentration of the culture medium was reduced to 0.5% for 1 hr before the experiment.

Human pulmonary arterial endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) were purchased from Lonza (Allendale, NJ). Cells were grown in EGM-2 supplemented with fetal bovine serum (FBS) and cultured in an incubator at 37°C in 5% CO2 and 95% air. Cells from passages 5 to 9 were used in the experiments.

HUVECs (Gibco, C0155C) and HLMVECs (Lonza, CC-2527) were used in our studies. Identity of endothelial cells was confirmed by immunostaining of CD31, von Williebrand Factor VIII, and positive for acetyated low density lipoprotein uptake. Cells were cultured in complete endothelial cell medium (ScienCell, 1001) and discarded after three passages. HEK-293T (ATCC, CRL-3216), EA.hy926 (ATCC, CRL-2922), and HEK-293 (ATCC, CRL-1573) were maintained in DMEM medium (Corning, 10013CV), and THP-1 cells (ATCC, TIB-202) were cultures in RPMI-1640 medium (Corning, 10–040 CM). All medium was supplemented with fetal bovine serum (FBS; Gibco, 1082147) and penicillin/streptomycin (Corning, 30–002 CI). Cell identities were confirmed at the commercial source (ATCC) using satellite tandem repeat profiling and were tested to be free from mycoplasma.