Journal: Nature Communications

Article Title: Oxidized mitochondrial DNA induces gasdermin D oligomerization in systemic lupus erythematosus

doi: 10.1038/s41467-023-36522-z

Figure Lengend Snippet: a Immunoblotting of GSDMD and GSDMD-N in BM neutrophils after treatment with IFN-γ, NS from WT mice, LS from lupus mice, or IFN-γ + LS for 12 h. b Quantitative analysis of GSDMD-N/GAPDH. c Immunoblotting of GSDMD and GSDMD-N in peripheral blood neutrophils of HV. Cells were pretreated with IFN-γ. Then the cells were added with NS form HV or LS from SLE patients for 12 h. d Quantitative analysis of GSDMD-N/GAPDH levels. e Immunoblotting for GSDMD and GSDMD-N in BM neutrophils from Caspase-1 − / − , Caspase-11 − / − and Caspase-1 − / − Caspase-11 − / − mice treated with IFN-γ + LS. f Quantitative analysis of GSDMD-N/GAPDH levels. g Immunoblotting of Serpinb1, Caspase-1, Caspase-11, and GSDMD levels in BM neutrophils treated with IFN-γ + LS, or IgG deleted LS for 12 h. h Quantitative analysis of Serpinb1, Caspase-1, Caspase-11, and GSDMD-N levels. i Immunoblotting analysis of Serpinb1, Caspase-1, Caspase-11, and GSDMD levels in WT or FcγR − / − BM neutrophils after IFN-γ + LS treatment for 12 h. j Quantitative analysis of Serpinb1, Caspase-1, and Caspase-11 levels. k Immunoblotting analysis of Serpinb1, Caspase-1, and Caspase-4 levels in neutrophils isolated from the peripheral blood of HV and SLE patients. l Quantitative analysis of Serpinb1, Caspase-1, and Caspase-4 levels. m Immunoblotting analysis of Serpinb1, Caspase-1, and Caspase-11 levels in the BM neutrophils from saline or PIL mice. n Quantitative analysis of Serpinb1, Caspase-1, and Caspase-11 levels. n = 6 mice ( b , f , h , j , n ) or 6 donors ( d , l ). The immunoblotting samples shown are from the same experiment. Two blots were processed in parallel ( d , l , n ). Three blots were processed in parallel ( b , f , h , j ). Data are shown as mean ± SD. Significance was examined by one-way ANOVA ( b , f , h , j ), or unpaired two-sided Student’s t test ( d , l , n ).

Article Snippet: Casp1 −/− mice were obtained from Cyagen biotechnology (S-KO-01331).

Techniques: Western Blot, Isolation, Saline

Journal: Nature Communications

Article Title: Oxidized mitochondrial DNA induces gasdermin D oligomerization in systemic lupus erythematosus

doi: 10.1038/s41467-023-36522-z

Figure Lengend Snippet: a Images of spleen from MRL/lpr mice or MRL/lpr with DSF treatment. Scale bar, 1 cm. b Quantitative analysis of spleen weight. c Images of lymph gland from MRL/lpr mice or MRL/lpr with DSF treatment. Scale bar, 1 cm. d Quantitative analysis of the weight of lymph gland (4 lymph glands). ELISA analysis of the levels of anti-dsDNA ( e ), anti-RNPs ( f ), anti-Sm ( g ), IL-1β ( h ) and IL-18 ( i ) in serum samples from the indicated groups. j qPCR analysis of D-loop in serum from indicated groups. k H&E staining of kidney section from indicated groups; scale bar = 100 μm. l Quantitative analysis of numbers of glomerular crescents from indicated groups. m ELISA of urine albumin-to-creatine ratio in MRL/mpj, MRL/lpr, and MRL/lpr mice treated with DSF. n Renal section from the indicated treatment groups were stained with or IgG (green) and C3 (red). Scale bar, 50 μm. o , p Quantitative analysis of the intensity of IgG and C3 per FOV. q Working model. ICs and IFN-γ in the serum of SLE patients promote the activation of GSDMD through serpinb1/caspase-1/11 pathway. Moreover, ICs also promote mitochondrial stress, which facilitates the release of Ox-mtDNA into the cytosol. Cytosolic Ox-mtDNA directly binds with GSDMD-N to promote GSDMD-N oligomerization and GSDMD pore formation. The subsequent extracellular NETs and mtDNA promote SLE pathogenesis. n = 6 mice, 2 independent experiments ( b , d – j , l , m , o , p ). Data are shown as means ± SD. Significance was examined with unpaired two-sided Student’s t test ( b , d ) one-way ANOVA ( e – j , l , m , o , p ).

Article Snippet: Casp1 −/− mice were obtained from Cyagen biotechnology (S-KO-01331).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Activation Assay