Fig. 7: TNF Complex IIb/caspase-8/caspase-3/GSDME-mediated pyroptosis is involved in TNF-α-induced sarcopenia in myotubes.
From: TNF-α contributes to sarcopenia through caspase-8/caspase-3/GSDME-mediated pyroptosis
A immunoblot of myosin heavy chain 1 (MHC1) in myotubes treated with different concentrations (0, 1, 10, or 100 ng/ml) of TNF-α for 72 h. Relative expression levels of MHC1 normalized to β-Tubulin based on densitometric analysis of immunoblots. B immunoblots of MHC1 in myotubes treated with TNF-α (100 ng/ml) +TAK1i (1 μM) at the indicated time points. Relative expression level of MHC1 normalized to GAPDH based on densitometric analysis of immunoblot. *P < 0.05 vs. 0 h. C immunoblots of MHC1 in myotubes treated with TNF-α (100 ng/ml) + TAK1i (1 μM) for 24 h. Relative expression level of MHC1 normalized to GAPDH based on densitometric analysis of immunoblot. D myotubes were pretreated with GSK'872 (1 μM) for 30 min and then Z-IETD-FMK (50 μM) for 1 h followed by treatment with TNF-α (100 ng/ml) for 72 h. The expression of MHC1 in myotubes were immunoblotted and analyzed based on densitometric analysis of immunoblots. E myotubes were pretreated with Z-DEVD-FMK (50 μM) for 1 h followed by treatment with TNF-α (100 ng/ml) for 72 h. The expressions of MHC1 in myotubes were immunoblotted and analyzed based on densitometric analysis of immunoblots. F–J myoblasts were transfected with GSDME knockdown lentivirus (LV-shGSDME) and then induced to differentiate into myotubes. Knockdown efficiency was examined by immunoblotting of GSDME (F). Morphologies of myoblasts and myotubes were observed by fluorescence microscopy (G). Scale bar: 100 μm. After treating myotubes with TNF-α (100 ng/ml) +TAK1i (1 μM) for 6 h, immunoblots of GSDME and GSDME-N (H) and detection of LDH release (J) in myotubes were performed. The expressions of MHC1 in myotubes treated with TNF-α (100 ng/ml) + TAK1i (1 μM) for 24 h were immunoblotted and analyzed based on densitometric analysis of immunoblots (I). Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significance. A–H n = 5 independent experiments. I, J n = 4 independent experiments.
