Figure 5

The pro-myogenic effect of insulin-like growth factor-1 depends on sphingosine kinase-1 and sphingosine kinase-2 engagement. (A) C2C12 myoblasts, transfected with scrambled (SCR) or sphingosine kinase (SK)1-siRNA were challenged with 50 ng/ml insulin-like growth factor-1 (IGF-1) for the last 24 h of transfection. Western analysis of SK1 and skeletal muscle marker proteins were 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. The histograms represent band intensity of SK1, 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). (B) Myoblasts transfected with SCR- or SK2-siRNA were treated and used as described in A. SK silencing in SK1- and SK2-siRNA transfected cells versus SCR-siRNA transfected cells was statistically significant (*P < 0.05). The effect of SK downregulation on IGF-1-induced expression of myogenin and caveolin-3 in IGF-1-challenged, SK-siRNA transfected cells versus control (IGF-1 added, SCR-siRNA transfection) was statistically significant by Student’s t test (#P < 0.05).