Figure 7
Role of sphingosine kinase in the mitogenic effect of insulin-like growth factor-1. (A) Serum-starved C2C12 myoblasts were pre-incubated for 30 min in the presence or absence of 1 μM SKI-2 before being 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 in untreated cells was 29893 ± 1584 dpm. Results are reported as -fold change over control (vehicle, no addition; set as 1). Data are mean ± SEM of at least three independent experiments performed in triplicate. The mitogenic effect of IGF-1 in challenged versus unchallenged cells was statistically significant by Student’s t test (*P < 0.05). The effect of SK inhibition on the mitogenic effect of IGF-1 in challenged cells versus control (no SKI-2, IGF-1 added) was statistically significant by Student’s t test (#P < 0.05). (B) Scrambled (SCR) or specific SK1- (upper panel) or SK2-siRNA (lower panel) transfected C2C12 cells 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 20781 ± 785 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 four independent experiments performed in triplicate. The effect of SK downregulation on IGF-1-induced [3 H]Thymidine incorporation in IGF-1-challenged, SK1- and SK2-siRNA transfected cells versus control (IGF-1 added, SCR-siRNA transfection) was statistically significant by Student's t test (#P < 0.05). Insert: cell extracts from C2C12 myoblasts transfected with SCR-, SK1-siRNA or SK2-siRNA were employed for Western analysis using anti-SK1 or anti-SK2 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 SK1 or SK2 was normalized to β-actin and reported as -fold change over control (SCR siRNA, no addition; set as 1).