Figure 2

S1P activates NF-κB through G protein coupled receptors. A. The upper panel shows the presence of mRNAs encoding G protein coupled S1P receptors in HeLa cells. + and - denote whether the extracted RNA was reverse transcribed or not. The lower panel shows Western blots of the S1P receptors in HeLa cells stimulated ± 3 μM S1P for 30 minutes. B. Concentration-response curve for S1P-induced activation of NF-κB (p65). HeLa cells were stimulated with varying concentrations of S1P for 30 minutes. Protein extracts were analyzed by NF-κB (p65) ELISA. 5 μg of protein was used from each extract. C. HeLa cells were preincubated with 50 ng/ml PTX for 16 h, 10 μM VPC23019 for 30 minutes or 10 μM JTE013 for 30 minutes prior to stimulation with 3 μM S1P for 30 minutes (left panel), or treated with S1P₂ or S1P₅ siRNA for 48 h prior to S1P stimulation. Proteins were then extracted and NF-κB (p65)-activation was assayed by ELISA. D. HeLa cells were stimulated with either vehicle, 3 μM S1P or 3 μM dihydro-S1P for 30 minutes. 5 μg of protein from each extract was used for the NF-κB (p65) ELISA. E. HeLa cells were pre-incubated with either vehicle, 10 μM DMS or 10 μM SKi for 5 minutes. The cells were then stimulated with 3 μM S1P for 10 minutes in the presence of [³H]sphingosine. The bars show synthesized cellular [³H]S1P as percent of the unstimulated control. F. The cells were pre-incubated either with vehicle, 10 μM sphingosine, 10 μM DMS or 10 μM SKi for 5 minutes. Following a 30-minute stimulation with 3 μM S1P, the cells were harvested, lyzed and the DNA-binding activity of p65 was measured from 5 μg of protein extract. The data points and bars in panels B-F denote the mean ± SEM of at least three independent experiments (*, p < 0.05).