Fig. 3
S1P signals through Gα12/13 and RhoA to induce COX-2 expression. a COX-2 protein expression was measured in WT cells pretreated with C3 exoenzyme (0.5 μg/mL) for 4 h prior to vehicle or S1P (0.5 μM) treatment for 6 h. COX-2 was normalized to GAPDH and expressed relative to the averaged ± inhibitor controls. Representative Western blot and data shown are mean ± SEM from three independent experiments run in triplicate. The blot represents a single gel where unnecessary lanes have been removed. **p < 0.01 between vehicle and S1P-treated groups and ##p < 0.01 between S1P-treated ± inhibitor groups. b Quantification and Western blot of COX-2 protein levels after knockdown of Gα12/13 with siRNA (2 μM) followed by S1P treatment (5 μM, 6 h). The blot represents a single gel where unnecessary lanes have been removed. Data shown are mean ± SEM of values from four independent experiments run in triplicate. COX-2 was normalized to GAPDH and expressed relative to the siRNA control. mRNA expression levels of COX-2 (c), IL-6 (d), and VEGFa (e) were measured by q-PCR following knockdown of Gα12/13 with siRNA (2 μM) and S1P treatment (0.5 μM, 1 h). Data shown are mean ± SEM from three independent experiments run in triplicate. COX-2, IL-6, and VEGFa were normalized to GAPDH and fold increase expressed relative to the vehicle-treated control siRNA. *p < 0.05 and **p < 0.01 between vehicle and S1P-treated groups and #p < 0.05 and ##p < 0.01 between control and Gα12/13 S1P-treated groups