Figure 4

Inhibition of human skeletal muscle cell (HuSKMC) differentiation by interleukin (IL)-1α and tumor necrosis factor (TNF)-α is mediated by the transforming growth factor-β-activated kinase (TAK)-1/p38/nuclear factor (NF)κB/Activin A/SMAD2/3 pathway. (A) Analysis of NFκB-luc assay (RGA) from HuSKMC myoblasts treated with IL-1α (1 ng/ml) and TNF-α (1 ng/ml) alone, and in combination with TAK-1 inhibitor (0.1-1 μmol/l) or withaferin A (0.1-1 μmol/l). Data are expressed as relative light units (RLU). Data are means ± SEM from four independent experiments. Differences from IL-1α- and TNF-α-treated HuSKMCs (first column),*P < 0.05. (B) Immunoblotting of phospho-TAK-1, phospho-SEK1/MKK4, phospho-p38MAPK, phospho-c-Jun, phospho-ATF2, phospho-NFκB, phospho-SMAD2 and phospho-SMAD3 of samples from HuSKMCs treated with TNF-α (0.1 to 10 ng/ml) or IL-1α (0.1 to 10 ng/ml) for 15 minutes alone, and in the presence of TAK-1 inhibitor (1 μmol/l) starting at the onset of differentiation. The TAK-1 inhibitor was given 3 hours before IL-1α or TNF-α stimulation. Shown are representative immunoblots. (C) Immunoblotting of phospho-SMAD2, phospho-SMAD3 and pAKT of samples from HuSKMCs treated with TNF-α (0.1 to 10 ng/ml) or IL-1α (0.1 to 10 ng/ml) for 24 hours, alone and in the presence of SB431542 (1 μmol/l), αActA (10 μg/ml), TAK-1 inhibitor (1 μmol/l), SB203580 (10 μmol/l) or withaferin A (300 nmol/l) starting at the onset of differentiation. Inhibitors were given 3 hours before IL-1α or TNF-α stimulation. Shown are representative immunoblots.