The pro-myogenic effect of insulin-like growth factor-1 depends on sphingosine kinase engagement. (A) C2C12 myoblasts were pre-incubated for 30 minutes with sphingosine kinase (SK) inhibitor (10 μM SKI-2) before being challenged with 50 ng/ml insulin-like growth factor (IGF-1) for the indicated time intervals. The content of myogenin and caveolin-3 (cav-3) was analyzed by Western blotting in cell lysates. Equally loaded protein was checked by expression of the non-muscle-specific β isoform of actin. A blot representative of four independent experiments with analogous results is shown. The histograms represent band intensity of myogenin and cav-3 normalized to β-actin and reported as mean ± SEM of four independent experiments, -fold change over control (time 24 h, no addition; set as 1). The effect of IGF-1 in challenged versus unchallenged cells at 24 h and 48 h on the expression levels of the myogenic markers was statistically significant by Student’s t test (*P < 0.05). The effect of SK inhibition on basal expression levels of the myogenic markers in SKI-2 treated versus SKI-2 untreated cells was statistically significant by Student’s t test (**P < 0.05). The effect of SK inhibition on IGF-1-induced expression of myogenin and cav-3 in challenged cells (IGF-1 and SKI-2 added) versus control (IGF-1 added, no SKI-2) was statistically significant by Student’s t test (#P < 0.05). (B) C2C12 cells were seeded on microscope slides and treated as described in A. Representative immunofluorescence images of C2C12 myoblasts treated for 72 h with 50 ng/ml IGF-1 stained with anti-myosin heavy chain (MHC) antibody and propidium iodide are shown. Fusion index and differentiation index were calculated as described in the Methods section. Data are mean ± SEM of three independent experiments. The effect of IGF-1 in challenged versus unchallenged cells on fusion index and differentiation index was statistically significant by Student’s t test (*P < 0.05); fusion index and differentiation index in IGF-1-challenged cells were significantly reduced by SKI-2 pre-incubation (Student’s t test #P < 0.05).