Figure 6

Inhibitors of ceramide synthesis upregulate anti-atrophic signaling pathways. (A) Myotubes were treated for 3 days with or without tumor necrosis factor (TNF)-α, in the presence of 100 nmol/l myriocin or 10 μmol/l GW4869, or both. (Left panel) Phospholipase (PL)D1 mRNA levels were measured by reverse transcription quantitative PCR, and normalized to the TATA box binding protein mRNA. Results are the mean ± SE of 3 to 6 measurements in duplicate. *Different from TNF-α alone: P = 0.05, **P < 0.001; different from control: P≤ 0.05. (Right panel) PLD1 protein in cell extracts was analyzed by western blotting. Results were normalized to the amount of tubulin, and are the mean ± SE of three experiments. **Different from control: P < 0.01; +different from TNF-α alone: P = 0.02. (B) Myotubes were treated for 3 days with or without TNF-α, in the presence of 100 nmol/l myriocin. (Left panel) S6K1 mRNA levels were measured by reverse transcription quantitative PCR and normalized to the TATA box binding protein mRNA. Results are the mean ± SE of three measurements in duplicate. **Different from both control and TNF-α alone: P < 0.01. (Right panel) Total S6K1 protein and phospho-Thr389 S6K1 in cell extracts were analyzed by western blotting. Results were normalized to the amount of tubulin, and are the mean ± SE of three determinations. +Different from control: P < 0.05; ++P < 0.02; **different from both control and TNF-α alone: P < 0.02. (C) Myotubes were treated for 3 days with or without TNF-α, in the presence of 100 nmol/l myriocin or 10 μmol/l GW4869. Ph-Ser473 Akt was analyzed by western blotting. Results were normalized to the amount of (left panel) tubulin or (right panel) total Akt, and are the mean ± SE of 3 to 5 determinations. NS, not significantly different from control. ***Different from TNF-α: P < 0.001, **P < 0.01, *P < 0.05.