Fig. 3: TNF-α induced GSDME-mediated pyroptosis in C2C12 myotubes.

A Morphology of C2C12 myoblasts and myotubes. Scale bar:100 μm. B immunofluorescence staining of myoblasts and myotubes for myosin heavy chain 1 (MHC1, green). Nuclei are stained with DAPI. Scale bar: 50 μm. C representative microscopic images were taken after myotubes were treated with TNF-α (100 ng/ml) at the indicated time points. Red arrows indicate cell death. Scale bar: 100 μm. D TEM micrographs were taken after myotubes were treated with TNF-α (100 ng/ml) for 72 h. Scale bar: 2.0μm. black triangles indicate cell membrane, white triangles indicate myofilaments, and asterisks indicate nucleus. E immunoblots of GSDME and GSDME-N in myotubes treated with TNF-α (100 ng/ml) at the indicated time points. The relative expression level of GSDME-N normalized to β-actin based on densitometric analysis of immunoblot. *P < 0.05 vs. 0 h, ^#P < 0.05 vs. 48 h. F, G immunoblots of GSDME and GSDME-N in myotubes treated with different concentrations (0, 1, 10, or 100 ng/ml) of TNF-α for 48 h (F) or 72 h (G). Relative expression levels of GSDME-N normalized to β-actin based on densitometric analysis of immunoblots. H, I cell death was determined by staining with Hoechst 33342/ propidium iodide (PI) (H) and measuring lactate dehydrogenase (LDH) release into the cell culture supernatant (I) after myotubes were treated with different concentrations (0, 1, 10 or 100 ng/ml) of TNF-α for 72 h. Scale bar: 50 μm. J Quantitation of the positive ratio of PI. Data are expressed as mean ± SD. **P < 0.01, ***P < 0.001, ns: no significance. n = 5 independent experiments.