produced and used to transduce HUVEC cells to express the miRNA molecules. Cell culture and retroviral transduction All cells were cultured in a humidified tissue culture incubator with 5% CO2 and 95% air at 37uC. Primary HUVECs were purchased from Lonza (Walkersville, MD, previously known as Clonetics) and grown in endothelial cell growth medium 2 (EGM- 2) (Lonza) as previously described [15]. U937 monocytic cell line was originally from American Type Culture Collection (ATCC, Manassas, VA). U937 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen or HyClone, Logan, UT). To prepare retroviral particles for cell transduction, the MSCV or pQCXIP retroviral construct was co-transfected with the amphotropic packaging vector pCL-10A1 (Imgenex, CA) into HEK 293T cells (ATCC, Manassas, VA) to make replication- deficient viral particles, which were used to transduce HUVEC cells to express target genes. HUVECs stably expressing the MSCV-IRES-GFP, MSCV-huGPR4-IRES-GFP, or MSCV- huGPR4 R115A-IRES-GFP construct were isolated by fluores- cence-activated cell sorting (FACS) based on green fluorescent protein (GFP) signals. HUVECs transduced with the pQCXIP or pQCXIP-N-Epac construct were selected with 2 mg/ml puromy- cin to enrich cells with the expression vector. Cell adhesion assay The U937-HUVEC cell adhesion assay was performed as previously described [42,43] with minor modifications. This approach examines the adhesion of U937 monocytic cells to a HUVEC monolayer under a static condition with no flow. Primary HUVECs from passage 3 to 7 were used for the cell adhesion assay. For isocapnic pH medium preparation, the pH buffering of cell culture media were carried out as previously described [15,19]. Briefly, EGM-2 media were buffered with 7.5 mM HEPES, 7.5 mM EPPS and 7.5 mM MES (named as HEM), and the pH was adjusted using NaOH or HCl and measured with a pH meter (Fisher). To prepare hypercapnic pH media, EGM-2 media were added to cell culture plates and incubated in humidified tissue culture incubators filled with ambient air, 5% CO2 or 20% CO2, respectively. The pH of the EGM-2 media pre-treated under these conditions was measured to be around 8.4, 7.4 and 6.4, respectively. HUVECs stably expressing the MSCV-IRES-GFP vector, MSCV-huGPR4- IRES-GFP, MSCV-huGPR4 R115A-IRES-GFP, GPR4 miRNAs or control miRNA were cultured in a 24-well plate with EGM-2 media to form a confluent monolayer. For isocapnic pH treatment, HUVECs were incubated in the EGM-2/HEM media at various pHs for 5 h to 15 h (overnight) in a regular incubator with 5% CO2. For hypercapnic pH treatment, HUVECs were treated with CO2-buffered EGM-2 pH media for 5 h in incubators with ambient air, 5% CO2 or 20% CO2, respectively. After the pH treatment, HUVEC cells were washed once with DMEM at pH 7.4. To exclude the effects of acidosis on U937 monocytic cells, the step of U937 cell attachment to HUVEC cells was carried out at pH 7.4. U937 cells were resuspended in DMEM/ HEM media with 10% FBS at pH 7.4, added at a density of 66104 cells/well in 1 ml of medium to a 24-well plate with HUVEC monolayer, and incubated for 1 h to allow cell attachment. Wells of the plate were gently washed with warm DMEM medium containing 10% FBS for at least 5 times to remove non-adherent U937 cells. Attached U937 cells from three random fields (excluding the edge of the wells which tended to have non-specific cell binding) were counted under an inverted Acidosis/GPR4 and Endothelial Cell Adhesion microscope with a 106 objective (total 1006 magnification) and the number of U937 cells per field was used as a functional readout to measure the adhesion capacity of HUVECs. To investigate the signaling pathways and adhesion molecules involved in acidosis/GPR4-induced cell adhesion, HUVECs were treated with various agents and blocking antibodies. The details of treatment conditions were described in the figure legends and/or in the results section. cAMP assay The production of cAMP in HUVECs was measured using the Amersham cAMP Biotrak Enzymeimmunoassay (EIA) kit from GE Healthcare (Piscataway, NJ) following the manufacturer’s instruction. Briefly, 2.56104 HUVEC cells/well were cultured in a 96-well plate and treated with 200 ml/well of EGM-2/HEM media or with CO2-buffered EGM-2 pH media at various pHs in the presence of 0.5 mM IBMX for 15 to 20 min. After the media were removed, 200 ml/well of lysis reagent 1B was immediately added to break cell membranes and release intracellular cAMP. 100 ml of cell lysate was transferred to a well of a donkey anti- rabbit Ig-coated plate. 100 ml of rabbit anti-cAMP antiserum was added and incubated for 2 h on ice, followed by the addition of 50 ml of cAMP-peroxidase conjugate and incubation for 1 h on ice. After all wells were washed with 400 ml of wash buffer four times, 150 ml of enzyme substrate was added into each well and incubated for 30 min at room temperature. The reaction was stopped by adding 100 ml/well of 1 M sulphuric acid, and the optical density was determined at 450 nm using a microplate reader (Molecular Devices, CA). The total intracellular cAMP was calculated according to the cAMP standard measured in parallel. Real-time RT-PCR (reverse transcriptase-polymerase chain reaction) Human GPR4 gene expression was measured in HUVECs that were transduced with the control vector, GPR4 or miRNAs and grown in regular EGM-2 medium. The gene expression of endothelial adhesion molecules was assessed in HUVECs that were transduced with the control vector, GPR4 or GPR4 R115A mutant and treated with EGM-2/HEM media at pH 8.4, 7.4, and 6.4 or CO2-buffered EGM-2 pH media for 5 h. Total RNAs were extracted from HUVECs using the RNeasy Plus kit (QIAGEN, MD), and were reverse transcribed using the SuperScript II reverse transcriptase (Invitrogen, CA). Real-time PCR reagents were purchased from Applied Biosystems Inc (ABI, Foster City, CA). Primers specific for the adhesion molecules were the TaqMan Gene Expression Assays from ABI: VCAM-1, Hs01003372_ml; E- selectin, Hs00174057_ml; ICAM-1, Hs99999152_ml; P-selectin, Hs00174583_m1. The sequences of human GPR4 primers were: (sense) 59-TTCGAGGAGCGCGTCTTT-39, (antisense) 59- GGTCCGCCACACAGTTGA-39, and probe FAM-59- CTGCA- TACCACAGCTCACTGGCTTTCAC -39-TAMRA as previous- ly described [44]. Human glyceraldehydes-3-phophate dehydroge- nase (GAPDH) TaqMan Gene Expression Assay (ABI, Cat. # 4333764T) was used as the internal control. Real-time PCR was performed in duplicate with a program of 50uC for 2 min, 95uC for 10 min followed by 40 cycles of 95uC for 15 s and 60uC for 1 min, and the data was acquired and analyzed using the ABI 7300-HT real-time PCR thermocycler. Western blot analysis HUVECs transduced with the myc-tagged N-Epac construct or the control pQCXIP vector were lysed in ice-cold RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X- PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e27586