Figure 6
Role of sphingosine 1-phosphate receptors in insulin-like growth factor-1-induced differentiation of mouse myoblasts. C2C12 myoblasts, transfected with scrambled (SCR) or with specific siRNA for individual sphingosine 1-phosphate receptors (S1PR), incubated in the absence (−) or in the presence (+) of 50 ng/ml insulin-like growth factor-1 (IGF-1) for the last 24 h of transfection, were checked for downregulation by real-time PCR (left). Middle, Western analysis of skeletal muscle marker proteins was performed in cell lysates. Equally loaded protein was checked by expression of the non-muscle-specific β isoform of actin. A blot representative of three independent experiments with analogous results is shown. Right, densitometric analysis. The histograms represent band intensity of myogenin and caveolin-3 (cav-3) normalized to β-actin and reported as mean ± SEM of three independent experiments, -fold change over control (time 24 h, no addition; set as 1). The effect of S1P2 downregulation on IGF-1-induced expression of myogenin and cav-3 in IGF-1-challenged, S1P2-siRNA transfected cells versus IGF-1-challenged, SCR-siRNA transfected cells was statistically significant by Student’s t test (#P < 0.05); S1PR silencing was statistically significant in S1PR-siRNA transfected cells versus SCR-transfected cells (*P < 0.05).