Figure 2

The negative effects of tumor necrosis factor (TNF)-α on L6 myotubes are counteracted by ceramide-synthesis inhibition. (A) Myotube area was measured after 3 days in the presence or absence of 15 ng/ml TNF-α, with the addition of 100 nmol/l myriocin, 10 μmol/l GW4869, or 1 μmol/l 3-0-methylsphingomyelin (OMS), or with myriocin plus either GW4869 or OMS. Shown are the mean ± SE of 5 to 9 experiments, with 10 fields considered for each condition. ***Different from all the other conditions: P ≤ 0.001. (B) ELISA quantification of the MHC content of myotubes treated for 3 days with 15 ng/ml TNF-α, with or without 100 nmol/l myriocin. The means ± SE of three measurements expressed as a percentage of control values are shown. ***Different from the other conditions: P ≤ 0.005. (C) Western blot analysis of myosin light chains (MLC) 1 and 3 from myotubes treated for 3 days with 15 ng/ml TNF-α, with or without 100 nmol/l myriocin. The diagram shows the quantification of MLC1 and MLC3 levels, after normalization by tubulin levels. Shown are the mean ± SE of three determinations. **Different from control and from TNF-α: P ≤ 0.002; *different from control: P ≤ 0.05. (D) Creatine kinase activity of myotubes treated or not by TNF-α, in the presence of 100 nmol/l myriocin, or 10 μmol/l GW4869, or 1 μmol/l OMS, or both myriocin and GW4869 or OMS. Shown are the mean ± SE of three determinations made in triplicate. ***Different from all the other conditions: P ≤ 0.001.