Fig. 3.

The effect of downregulation of cytosolic phospholipase A₂α (cPLA₂α) or iPLA₂ on S1P-induced AA release. Downregulation of cPLA₂α or iPLA₂ by small interfering RNAs (siRNAs) was performed as described in materials and methods, and cells were stimulated with 500 nM S1P for 10 min. The AA release was represented as fold change compared with the vehicle-stimulated control cells (A, top). Forty-eight hours after siRNA transfection, cells were harvested, and cPLA₂α and iPLA₂ mRNA expression levels were analyzed by real-time PCR TaqMan analysis (A, bottom) and Western blot analysis (B). The data represent the means ± SD of 6 samples from a single experiment representative of 3 independent experiments, *P < 0.01 by Student's t-test comparing S1P-treated knockdown cells and control siRNA-treated cells. Western blot analysis was consistent across 3 independent experiments. Real-time PCRs were done in triplicate in 2 independent experiments.