out at room temperature. Briefly, HUVEC/Vector and HUVEC/ GPR4 cells were seeded in 8-well chamber slides, and grown overnight to allow cells to attach. Cells were then treated with EGM-2/HEM media at pH 8.4, 7.4, and 6.4 for 5 h, or with EGM-2 medium containing 10 nM thrombin for 20 min (for P- selectin only). After the treatment, cells were washed once with PBS and fixed with 4% paraformaldehyde in PBS for 15 min. Cells were then washed three times with PBS, permeabilized with PBST (PBS with 0.1% Tween 20) for 4 min and incubated with the blocking solution consisting of 5% normal goat serum in PBST for 30 min. For P-selectin staining, cells were fixed with 100% methanol at 220uC for 10 min, without the need for additional cell permeabilization. Afterwards, mouse monoclonal primary antibodies diluted in the blocking solution, including E-selectin antibody, VCAM-1 antibody, ICAM-1 antibody, or P-selectin antibody (R&D Systems), were applied to the cells at the concentration of 10 mg/ml and incubated for 1 h. After several washes with PBST, cells were incubated with Rhodamine Red- conjugated goat anti-mouse IgG secondary antibody (1:200) (Invitrogen, R6393) for 1 h. Thereafter, unbound antibodies were removed by several washes with PBS, and cells were mounted in the anti-fade mounting medium (Vector Laboratories) and examined under a fluorescence microscope (Nikon) connected with a digital camera (Zeiss). Statistical analysis Data were analyzed using the GraphPad Prism 5 software (GraphPad Software, Inc., CA) and depicted as mean 6 SEM. P,0.05 was considered statistically significant. Results Activation of GPR4 by isocapnic acidosis increases HUVEC cell adhesion Primary HUVECs, which have endogenous GPR4 expression, were used as a model system to study the effects of acidosis/ GPR4 signaling on endothelial cell adhesion. HUVECs were stably transduced with the MSCV-IRES-GFP retroviral control vector (designated as HUVEC/Vector cells) or the MSCV- huGPR4-IRES-GFP construct (designated as HUVEC/GPR4 cells). To examine the adhesiveness of HUVEC cells and to model the interaction between endothelium and blood cells, we Acidosis/GPR4 and Endothelial Cell Adhesion utilized the static HUVEC-U937 monocyte attachment assay as a functional readout [42,43]. After non-adherent cells were removed by several washes, the U937 cells attached to the flat HUVEC monolayer could be readily detected as small, round and reflectile cells under an inverted microscope (Fig. 1A). For pH treatment, EGM-2 media were buffered with the chemicals HEPES, EPPS and MES (collectively as HEM) and the pH was adjusted with HCl or NaOH to generate isocapnic acidosis or alkalosis. When HUVEC/Vector cells were treated with pH 8.2, 7.8 and 7.4 (6.3 nM, 15.8 nM, and 40 nM H+, respectively), the cells exhibited a low capacity to adhere U937 monocytes. The treatment of pH 7.0 and 6.6 (100 nM and 251 nM H+, respectively) increased HUVEC/Vector cell adhesion by 2- to 4-fold (Fig. 1B). With the overexpression of human GPR4 in HUVEC/GPR4 cells, acidosis significantly augmented the adhesiveness of HUVEC cells by 5- to 10-fold (Fig. 1B). As measured by real-time RT-PCR, GPR4 was overexpressed about 12-fold in HUVEC/GPR4 cells when compared to HUVEC/Vector cells (Fig. 1C). Similarly, in HUVEC cells overexpressing the mouse GPR4-GFP fusion gene [15,19], a significant increase of HUVEC adhesiveness was also observed upon acidic pH treatment (Fig. 1D). U937 cells cultured in RPMI medium showed similar results as that from the cells cultured in DMEM medium (Fig. S1A). In addition to U937 monocytes, the adhesion of HUVECs with another type of leukocytes, the HL-60 promyelocytic cells (neutrophils), under the static condition was also increased when HUVECs were stimulated by acidosis/GPR4 (Fig. S1B). These results suggest that activation of GPR4 by acidosis induces an adhesive phenotype of HUVECs. To further investigate the role of GPR4 in endothelial cell adhesion induced by isocapnic acidosis, microRNAs-based short-hairpin RNA interference (miRNAs) was used to knock- down the endogenous GPR4 gene expression in HUVECs. We tested several miRNAs targeting different regions of GPR4 mRNA and identified two independent miRNAs that efficiently down-regulated GPR4 expression by more than 80% in HUVECs as measured by real-time RT-PCR (Fig. 1C). A negative control miRNA without sequence homology did not inhibit GPR4 expression. In the U937-HUVEC cell adhesion assay, acidosis increased the adhesiveness of HUVECs trans- duced with the negative control miRNA, but not HUVECs Figure 2. G protein signaling is indispensible for acidosis/GPR4-induced HUVEC adhesion. (A) HUVECs were transduced with GPR4 or the GPR4 R115A mutant expression vector. The intracellular cAMP level in HUVECs was determined by the cAMP assay as described under ‘‘Materials and Methods’’. The total intracellular cAMP was calculated according to the cAMP standard measured in parallel. *, P,0.05; ns, not significant (P.0.05). (B) HUVEC cells as described in (A) were treated with indicated pHs and the cell adhesion assay was performed as described under ‘‘Materials and Methods’’. ***, P,0.001. The results are representative of two independent experiments. Error bars are the mean 6 SEM. doi:10.1371/journal.pone.0027586.g002 PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27586