Fig 2: Activation of Casp-8 was required for TNF-a-induced pyroptosis in C2C12 myotubes.A immunoblots of Casp-8 and Cl-Casp-8 in myotubes treated with different concentrations (0, 1, 10, or 100 ng/ml) of TNF-a for 72 h. Relative expression levels of Cl-Casp-8 normalized to ß-Tubulin based on densitometric analysis of immunoblots. B–E 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-a (100 ng/ml) for 72 h. The expression of GSDME, GSDME-N, Casp-3, and Cl-Casp-3 in myotubes were immunoblotted and analyzed based on densitometric analysis of immunoblots (B). Cell death was determined by staining with Hoechst 33342/PI (C) and measuring LDH release into the cell culture supernatant (E) after myotubes treatments. Scale bar: 50 µm. D Quantitation of positive ratio of PI. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significance. n = 5 independent experiments.

Fig 3: TNF Complex IIb but not Complex IIa enables TNF-a-induced caspase-8 activation and subsequent caspase-3/GSDME-mediated pyroptosis occurrence.A, B immunoblots of GSDME and GSDME-N in myotubes treated with TNF-a (100 ng/ml) + TAK1 inhibitor (TAK1i, 1 µM) (A) or TNF-a (100 ng/ml) +cycloheximide (CHX, 10 µg/ml) (B) at the indicated time points. Relative expression level of GSDME-N normalized to GAPDH based on densitometric analysis of immunoblot. *P < 0.05 vs. 0 h. C, D immunoblots of GSDME, GSDME-N, Casp-8, and Cl-Casp-8 in myotubes treated with TNF-a (100 ng/ml) + TAK1i (1 µM) or TNF-a (100 ng/ml) + CHX (10 µg/ml) for 6 h (D) or 24 h (C). Relative expression level of GSDME-N and Cl-Casp-8 normalized to GAPDH based on densitometric analysis of immunoblot. E–H myotubes were treated with TNF-a (100 ng/ml) +TAK1i (1 µM) for 6 h. Cell death was determined by measuring LDH release into the cell culture supernatant (E) and staining with Hoechst 33342/PI (H) after myotubes treatments. Scale bar: 50 µm.The expressions of GSDME, GSDME-N, Casp-3, and Cl-Casp-3 in myotubes were immunoblotted and analyzed based on densitometric analysis of immunoblots (F). Representative optical microscope images and TEM micrographs were taken after myotubes were treated with or without TNF-a + TAK1i (G). Red arrows indicate cell death, black triangles indicate cell membrane, white triangles indicate myofilaments, and asterisks indicate nucleus. Scale bar: 100µm for optical microscope images. Scale bar: 2.0 µm for TEM micrographs. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significance. n = 5 independent experiments.

Fig 4: GSDME-mediated pyroptosis and activation of apoptosic caspase-8/-3 were present in GM of aged mice with sarcopenia.A Representative immunoblots of GSDMD, N terminus of GSDMD(GSDMD-N), GSDME, N terminus of GSDME(GSDME-N), caspase (Casp)-8, cleaved-Casp(Cl-Casp)-8, Casp-3 and Cl-Casp-3 in GM of mice at different ages. B, C Relative expression levels of GSDMD-N, GSDME-N, Cl-Casp-8, and Cl-Casp-3 normalized to GAPDH based on densitometric analysis of immunoblots. D TUNEL staining, immunofluorescence staining of GSDME and immunohistochemical staining of Cl-Casp-8 and Cl-Casp-3 in GM of mice at different ages. Scale bar: 50 µm. E positive rates of TUNEL staining in the mice at different ages. F quantitation of immunohistochemical analyses for Cl-Casp-8 and Cl-Casp-3. G, correlation analysis of the relative expression level of GSDME-N with relative grip strength and GMI in mice. Data are expressed as mean ± SD. *P < 0.05, ***P < 0.001, ns: no significance. n = 8 mice per group.