Fig. 5: Pharmacologically blocking mPTP ameliorated activation of the mtDNA-cGAS-STING-NLRP3 axis and pyroptosis in human NP cells.

Cyclosporine (CsA) or DMSO in combination with 100 μM TBHP was used to treat NP cells for 24 h. a Representative fluorescence images of calcein AM/Co²⁺ quencher staining and the quantitative analysis of fluorescence intensity for opening levels of mPTP in NP cells after CsA treatment compared to the controls. b Representative scatter plots of JC-1 staining and the quantitative analysis of MMP after CsA treatment compared to the vehicle control. c Representative TEM images showing mitochondrial morphology in the CsA- or DMSO-treated NP cells. d DNA agar gelatin electrophoresis images of mtDNAs in cytosolic or total lysates from the CsA- or DMSO-treated NP cells. e RT-qPCR quantitative analysis of cytosolic mtDNAs normalized to mtDNAs of total lysates from the CsA- or DMSO-treated NP cells. f Representative fluorescence images of dsDNA and mitochondria in the CsA- or DMSO-treated NP cells. g Representative western blotting images of cGAS and STING in the CsA- or DMSO-treated NP cells. h Quantification of the protein expression levels of cGAS and STING in the CsA-treated NP cells compared to the controls. i Representative immunofluorescence images of STING with different degrees of polymerization in the CsA- or DMSO-treated NP cells. j Representative DNA immunoprecipitation images in the CsA- or DMSO-treated NP cells. k Quantification of the enrichment level of cGAS in the DNA immunoprecipitates in the CsA-treated NP cells compared to the controls. l The physical interaction between dsDNA and cGAS from in situ PLA images in the CsA- or DMSO-treated NP cells. m Representative western blotting images of NLRP3, ASC, pro-CASP-1, cleaved CASP-1, GSDMD, and cleaved GSDMD in the CsA- or DMSO-treated NP cells. n Quantification of the protein levels of NLRP3 and ASC in the CsA-treated NP cells compared to the controls. o The ratio of cleaved CASP-1 to pro-CASP-1 in the CsA-treated NP cells compared to the controls. p The ratio of cleaved GSDMD to GSDMD in the CsA-treated NP cells compared to the controls. q The enzymatic activity of CASP-1 in the CsA- or TBHP-treated NP cells. r The quantitative level of IL-1β in cell culture supernatants after CsA or DMSO treatment. s TEM images of the CsA or DMSO-treated NP cells. t Representative fluorescence images of Hoechst/PI staining in the CsA- or DMSO-treated NP cells. Data are shown as the mean ± SD of at least three independent experiments. Statistical analyses were conducted using two-way ANOVA and Student’s t test. The P value is indicated by stars: *P < 0.05, **P < 0.01, ***P < 0.001.