Fig. 7. MtDNA directly interacts with GSDMD-N.
a qPCR analysis of mtDNA following GSDMD pulldown under the indicated treatment. BM neutrophils were treated with LPS + Nigericin + H2O2, LPS + Nigericin, or IFN-γ + LS. n = 6 mice. b Immunoblotting of TOMM20 and GSDMD. BM neutrophils were treated with IFN-γ + LS for 12 h, or pretreated with Mito-TEMPO. Lysates were co-immunoprecipitated with anti-GSDMD, and co-immunoprecipitates were then spotted on a nitrocellulose membrane, UV crosslinked, and probed with antibodies specific for 8OHdG, or were separated via SDS-PAGE for GSDMD and TOMM20. c Quantitative analysis of 8OHdG and TOMM20. n = 5 mice. The samples shown are from the same experiment. Three blots were processed in parallel. d Immunoblotting of TOMM20 and 8OHdG in peripheral blood neutrophils from HV or SLE patients (n = 6). e Quantitative analysis of 8OHdG and TOMM20. The samples shown are from the same experiment. Three blots were processed in parallel. Relative mtDNA enrichment was assessed via qPCR in indicated cells. 293T (f) and Gsdmd−/− MEF cells (g) were transfected with Flag-full-GSDMD, Flag-GSDMD-N or GSDMD-C, then treated with H2O2 (100 μM) for 4 h. n = 6 samples pooled from 6 independent experiments. h The cluster of four evolutionarily conserved, positively charged amino acids (red and underlined) in GSDMD-N were mutated to Ala. i X-ray crystal structure of the murine GSDMD (PDB: 6N9N). Dissociation constants (KD) of human GSDMD with human mtDNA (j) and Ox-mtDNA (k). KD of peptides from GSDMD-N with human mtDNA (l) and Ox-mtDNA (m). n, o Measurements of KD of mutant peptides from GSDMD-N with mtDNA (n) and Ox-mtDNA (o). The KD was derived from the binding response as a function of the His-tagged GSDMD or His-tagged peptides. Errors in KD represent fitting errors. Representative of three independent experiments (b, d, j–o). Data are presented as means ± SD. Significance was examined with one-way ANOVA (a, c, f, g) or unpaired two-sided Student’s t test (e).