Figure 3

Inhibition of human skeletal muscle cell (HuSKMC) differentiation by interleukin (IL)-1α and tumor necrosis factor (TNF)-α is mediated by transforming growth factor-β-activated kinase (TAK)-1/p38/nuclear factor (NF)κB/Activin A/SMAD2/3 pathway. (A) HuSKMC myotubes differentiated for 5 days in the absence (Con) and presence of IL-1α (0.1 ng/ml) or TNF-α (0.1 ng/ml), alone and in combination with the neutralizing neutralizing anti-Activin A antibody (10 μg/ml; αActA) or TAK-1 inhibitor (1 μmol/l) were stained with anti-myosin heavy chain (MyHC) and DAPI. Shown are representative pictures and analysis of fusion index, which was determined as the percentage of nuclei occurring in myotubes stained with MyHC on four pictures taken. Data are means ± SEM from four to six independent experiments. Differences from untreated HuSKMCs (control; first column),*P < 0.05; differences from IL-1α- and TNF-α-treated HuSKMCs (control, second and third column), #P < 0.05. (B) Analysis of CK activity from in HuSKMC myotubes differentiated for 4 days and treated with either IL-1α (0.01-0.1 ng/ml) and TNF-α alone (0.01-0.1 ng/ml), and in combination with αActA (10 μg/ml), TAK-1 inhibitor (1 μmol/l), SB203580 (10 μmol/l) or withaferin A (100 nmol/l). Data are expressed as percentage of control from untreated HuSKMCs. Data are means ± SEM from three to nine independent experiments. Differences from untreated HuSKMCs (control; first column),*P < 0.05; differences from IL-1α- and TNF-α-treated HuSKMCs (control, second to fourth columns), #P < 0.05. (C) Analysis of CK activity from in HuSKMC myotubes differentiated for 4 days and treated with IL-1α (0.01-0.1 ng/ml) and TNF-α alone (0.01-0.1 ng/ml) after treatment with either small interfering (si)RNA against non-targeting control (siNTC), siActivin A β-chain or siSMAD2/3. Data are means ± SEM from five to seven independent experiments. Differences from siNTC-treated HuSKMCs (siNTC; first column),*P < 0.05; differences from IL-1α- and TNF-α-treated HuSKMCs (siNTC, second to fourth columns), #P < 0.05.