Journal: Nature Communications
Article Title: NCX1 reverse mode promotes calcium-dependent Neutrophil Extracellular Trap formation and lung damage in chronic obstructive pulmonary disease
doi: 10.1038/s41467-026-69636-1
Figure Lengend Snippet: a Representative immunofluorescence images of NCX1 (purple) co-localization with different immune cell-type markers (CD3 + , T cells, CD68 + , macrophages, CD66b + , neutrophils; CD11c + , neutrophils/dendritic cells) in distal airways and bronchioles and adjacent alveolar regions from Mixed CBE patients ( n = 5 patients). Blue, DAPI-stained nuclei. Light blue indicates colocalization of NCX1 with neutrophils. Scale bar = 30 µm. b RT-qPCR analyzes mRNA levels of NCX1 in human lung tissue from Control patients ( n = 20), emphysema patients ( n = 10), and Mixed CBE patients ( n = 20). c Western blot analysis of NCX1 protein levels in lung tissues from Control patients and Mixed CBE patients ( n = 10), normalized to β-actin and displayed relative to controls. Uncropped blots in Source Data. d , e RT-qPCR analyzes mRNA levels of MPO and NE in lung tissues from Control individuals ( n = 20), emphysema patients ( n = 10), and Mixed CBE patients ( n = 20). f–h Western blot analysis of MPO and NE expression in lung tissues from Control patients and Mixed CBE patients ( n = 10), normalized to β-actin and displayed relative to controls. Uncropped blots in Source Data. i , j Spearman’s correlation analyzes the correlation of NCX1 expression with MPO and NE levels. 95% CI: 0.5281 to 0.8724 for ( i ), 0.7029 to 1.007 for ( j ). k Representative immunofluorescence images show expression and localization of CD66b (green) and NCX1 (purple) in lung tissues of Control and Mixed CBE patients. Scale bar = 50 µm. Blue, DAPI-stained nuclei. l–n Flow cytometry identifies neutrophil numbers ( m ) and NCX1 mean fluorescence intensity ( n ) on neutrophils in human BALF ( n = 5 individuals). o Representative immunofluorescence images show expression of NCX1 in neutrophils from human BALF of Control and Mixed CBE patients. Scale bar = 2 µm. Blue, DAPI-stained nuclei. Data point represents one biologically independent replicate with three technical replicates ( b , d , e ), one biologically independent replicate with two technical replicates ( c , g , h ). Data points represent biologically independent replicates ( m , n ). Quantitative data are presented as min to max with all points mean ( b , d , e ), and as Mean ± SD ( c , g , h , m , n ). Two-sided t -test ( g , h , m , n ) and one-way ANOVA with Tukey’s multiple comparison test ( b , d , e ) were used to calculate the p- values. At least 3 times, each experiment was independently repeated with similar results. Source data are provided as a Source Data file. CBE chronic bronchitis and emphysema; MPO myeloperoxidase; NE neutrophil elastase; BALF bronchoalveolar lavage fluid; SCC-A forward scatter-area; FSC-H forward scatter-height; FSC-A side scatter-area; ns no significance.
Article Snippet: The samples were then incubated overnight at 4 °C with primary antibodies: CD11c (Servicebio, GB11059, 1:200), CD68 (Proteintech, 25747-1, 1:200), CD3 (Servicebio, GB11014, 1:100), Ly-6G (Invitrogen, 14-5931-82, 1:50), αSMA (Invitrogen, 50976082, 1:200), CD66b (Novus Biologicals, NB100-77808, 1:100), SFTPC (Proteintech, 10774-1-AP, 1:200), MPO (Proteintech, 22225-1-AP, 1:200), NE (Abclonal, A8953, 1:200), Cit-H3 (Abways, CY6587, 1:250) and NCX1 (Abclonal, A5583, 1:200).
Techniques: Immunofluorescence, Staining, Quantitative RT-PCR, Control, Western Blot, Expressing, Flow Cytometry, Fluorescence, Comparison
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