Fig. 6: TNF Complex IIb but not Complex IIa enables TNF-α-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-α (100 ng/ml) + TAK1 inhibitor (TAK1i, 1 μM) (A) or TNF-α (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-α (100 ng/ml) + TAK1i (1 μM) or TNF-α (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-α (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-α + 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.