Role of sphingosine 1-phosphate receptors in the mitogenic effect of insulin-like growth factor-1. (A) Quantitative mRNA analysis was performed in C2C12 myoblasts transfected with non-specific scrambled (SCR) siRNA or with siRNA specific for sphingosine 1-phosphate receptors (S1PR); the content of housekeeping gene 18 S rRNA was analyzed in parallel. Results are expressed as -fold changes according to the 2−ΔΔCT method, utilizing each receptor subtype in cells transfected with SCR-siRNA as a calibrator. Data are mean ± SEM of three independent experiments performed in triplicate. S1PR silencing in S1PR-siRNA transfected cells versus SCR-siRNA transfected cells was statistically significant (*P < 0.05). (B) Serum-starved C2C12 myoblasts transfected with unspecific SCR-siRNA, or with specific siRNA for individual S1PR, were stimulated with 50 ng/ml insulin-like growth factor 1 (IGF-1) for 16 h. [3 H]Thymidine (1 μCi/well) was added during the last hour of incubation. [3 H]Thymidine incorporation was 19643 ± 844 dpm in control cells (untreated SCR-transfected cells). Results are reported as -fold change over control (SCR siRNA, no addition; set as 1). Data are mean ± SEM of at least three independent experiments performed in triplicate. The effect of S1P1 and S1P3 downregulation on the IGF-1-induced [3H]Thymidine incorporation in IGF-1-challenged, S1P1- and S1P3-siRNA transfected cells versus IGF-1-challenged, SCR-siRNA transfected cells was statistically significant by Student's t test (#P < 0.05).