Fig. 3: IL-18 secretion and MCs pyroptosis by NLRP3 inflammasome activation.

A Representative images of FLICA co-staining with PI in nLung and LUAD MCs after stimulation with or without LPS and Nig using flow cytometry (upper). Cumulative data for active caspase-1 in nLung and LUAD MCs (lower). Data are presented as mean + SEM, the heights of bars present the mean value and the error bars represent the SEM value. Each group includes 6 replicates, and p values were determined by two-tailed paired Student’s t-test. FMO fluorescence minus one. B Representative images of FLICA (green) co-staining with ASC (yellow), CD117 (purple), and NLRP3 (red) in LUVA cell line using imaging flow cytometry. BF, bright-field. Scale bars, 10 μm. Each group includes more than 4 replicates. C The concentration of IL-18 in cell culture supernatants of MCs after stimulation with or without LPS and Nig in the presence or absence of indicated caspases inhibitors. Each group includes 6 replicates, p values were determined by two-tailed Student’s t-test. D Immunoblot analysis of cell lysates of nLung and LUAD MCs after stimulation with or without LPS and Nig in the presence or absence of indicated inhibitors. Each group includes more than 3 replicates. E Representative images of FLICA staining in nLung and LUAD MCs after stimulation with or without LPS and Nig using flow cytometry in the presence or absence of indicated inhibitors (left). Cumulative data of FLICA⁺ cells (right). Each group includes 5 replicates, p values were determined by two-tailed paired Student’s t-test. F Heatmap showing the expression of GSDM family genes in LUVA cell line, nLung MCs (nMC), and LUAD MCs (tMC). G Representative mIF staining of Asp275 (cleaved GSDMD, orange), and tryptase (blue) for MCs in LUAD sections. Nuclei were counterstained with DAPI (gray). Scale bars, 30 μm. Each group includes more than 3 replicates. H The concentration of IL-18 in cell culture supernatants of LUVA clones (including wild-type (WT), two GSDMD KO clones: KO#10 and KO#15) after stimulation with or without LPS and Nig by ELISA. Data are presented as mean + SEM, the heights of bars present the mean value and the error bars represent the SEM value. Each group includes 8 replicates, p values were determined by two-tailed Student’s t-test. I Quantification of dead cells on the basis of PI staining in WT or CRISPR–Cas9 KO LUVA clones. Data are presented as mean + SEM, the heights of bars present the mean value and the error bars represent the SEM value. Each group includes 6 replicates, p values were determined by two-tailed Student’s t-test. J CytoTox 96 Non-Radioactive Cytotoxicity Assay from WT or CRISPR–Cas9 KO LUVA clones. Each group includes 4 replicates, p values were determined by two-tailed Student’s t-test. K Representative flow images of PI staining in caspase 1⁺ or caspase 1^- MCs from nLung and LUAD (left). Cumulative data of PI⁺ cells in caspase 1⁺ or caspase 1^- MCs from nLung and LUAD (right). Data are presented as mean + SEM, the heights of bars present the mean value and the error bars represent the SEM value. Each group includes 4 replicates, p values were determined by two-tailed paired Student’s t-test. L CytoTox 96 Non-Radioactive Cytotoxicity Assay from LUVA cell line in the presence or absence of indicated NLRP3 inhibitors. Each group includes 8 replicates, p values were determined by two-tailed Student’s t-test. M CytoTox 96 Non-Radioactive Cytotoxicity Assay from LUVA cell line in the presence or absence of indicated caspase inhibitors. Each group includes 8 replicates, p values were determined by two-tailed Student’s t-test. See Supplementary Fig. 3.