Fig. 1: The upregulation of GSDMD and mROS in neutrophils from breast cancer metastatic mouse model.

a The diagram of animal model. b Fluorescence-activated Cell Sorting analysis of lung neutrophils from control and 4T1 mice, cells were sorted by flow cytometry (CD11b⁺Ly6G⁺). c Immunoblot analysis of GSDMD and GSDND-N of lung neutrophils sorted from control and 4T1 mice. d Non-reducing immunoblot analysis of GSDMD monomer and oligomer of lung neutrophils sorted from control and 4T1 mice. e Quantification of GSDMD (the ratio of GSDMD-N to GAPDH in (c), the ratio of GSDMD oligomer to GAPDH in (d). n = 3. f Transmission electron microscope images of mitochondria in neutrophil from lung of control and 4T1 bearing mice. Scale bar, 200 nm. g Immunofluorescence staining of GSDMD in cytosol and cell membrane (Did staining) in lung neutrophils from control and 4T1 bearing mice. Scale bar, 3 μm. h Quantitative analysis of colocalization of GSDMD and Did. i Histograms of Mito-Sox fluorescence intensity in neutrophils from control and 4T1 bearing mice analyzed by flow cytometry. j Quantification of mean fluorescence intensity (MFI) of Mito-Sox as indicated in (f). n = 3. k Relative expression of GSDMD-N/GAPDH in bone marrow neutrophils is positively correlated with Mito-Sox intensity in lung neutrophils from 4T1 bearing mice. l Relative expression of GSDMD-N/GAPDH in lung neutrophils is positively correlated with serum HMGB1 from 4T1 bearing mice. Data are presented as means ± SEM. Pearson’s correlation analysis for (k, l). Significance was examined with two-tailed unpaired Student’s t test (e) or Student’s t test (h, j). ^**P < 0.01, ^***P < 0.001.