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. Author manuscript; available in PMC: 2021 Mar 14.
Published in final edited form as: Nat Cell Biol. 2020 Sep 14;22(10):1264–1275. doi: 10.1038/s41556-020-0575-z

Fig. 6: GSDMC N-terminal domain (aa 1–365) binds to cell membrane and induces pyroptosis.

Fig. 6:

(a and b) Analysis of the pore-forming activity of caspase-8-cleaved GSDMC by high-resolution electron microscopy (a) and liposome leakage assay (b). Red arrows in (a) indicate pores. Triton X-100 was added at 20 min to achieve 100% leakage in (b). GSDMC-FL, full-length GSDMC. Scale bar, 100 nm. The experiments were repeated three times with similar results. (c) Membrane lipid-binding activity of GSDMC fragments, phosphatidylethanolamine (PE) as a negative control for liposomes. S, lipid-free supernatant; P, lipid-containing pellet; PI(4,5)P2, phosphatidylinositol-4,5-bisphosphate; GSDMC-N, N-terminal domain of GSDMC; GSDMC-C, C-terminal domain of GSDMC. The experiment was repeated three times with similar results. (d) Immunoblotting of wild-type (WT) and D365A-mutant (D365A) GSDMC proteins cleaved by caspase-8 with anti-His tag antibody. The experiment was repeated three times with similar results. (e) Fluorescent imaging of HeLa cells transfected with WT or D365A GSDMC genes and treated with TNFα plus CHX. Dying cells were stained with nucleic acid dye SYTOX green. Red arrows indicate cell swelling with large bubbles. Scale bar, 20 μm. The experiment was repeated four times with similar results. (f) LDH-released cell death is shown as mean ± SD of n = 3 biologically independent experiments. P values were determined by two-sided Student’s t-test. (g) Immunoblotting of GSDMC in HeLa cells expressing WT or D365A GSDMC and treated with TNFα plus CHX. The experiment was repeated three times with similar results. Statistical source data and unprocessed blots are provided in Source Data Fig. 6.