Figure 9
Sphingosine 1-phosphate lyase is not involved in insulin-like growth factor-1 biological action in C2C12 myoblasts. (A) C2C12 myoblasts, transfected with scrambled (SCR) or sphingosine 1-phosphate lyase (SPL)-siRNA, were 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. Western analysis of SPL and 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. The histograms represent band intensity of SPL, 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). SPL silencing in SPL-siRNA transfected cells versus SCR-siRNA transfected cells was statistically significant by Student’s t test (*P < 0.05). The effect of SPL downregulation on basal expression levels of myogenin and cav-3 in SPL-siRNA transfected, unchallenged cells versus SCR-siRNA transfected, unchallenged cells was statistically significant by Student’s t test (#P < 0.05). (B) Myoblasts transfected with unspecific SCR-siRNA or with specific SPL-siRNA were treated or not treated with 50 ng/ml IGF-1 for 16 h. [3 H]Thymidine (1 μCi/well) was added during the last hour of incubation. [3 H]Thymidine incorporation was 21781 ± 993 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. Insert: cell extracts from C2C12 myoblasts transfected with SCR- and SPL-siRNA were employed for Western analysis using anti-SPL antibodies. Equally loaded protein was checked by expression of β-actin. A blot representative of at least four independent experiments with analogous results is shown. Band intensity of SPL was normalized to β-actin and reported as -fold change over control (SCR siRNA, no addition; set as 1).