Fig. 7: NCX1 reverse-mode transport mediates Ca2+ overload and NETs release in response to CSE. | Nature Communications

Fig. 7: NCX1 reverse-mode transport mediates Ca2+ overload and NETs release in response to CSE.

From: NCX1 reverse mode promotes calcium-dependent Neutrophil Extracellular Trap formation and lung damage in chronic obstructive pulmonary disease

Fig. 7: NCX1 reverse-mode transport mediates Ca2+ overload and NETs release in response to CSE.The alternative text for this image may have been generated using AI.

a–c RT-qPCR and WB assess NCX1 expression in neutrophils upon CSE, fMLP and LPS stimulation (n = 3). d Immunofluorescence confirms NCX1 elevation indicated by Ly6g (green) and NCX1 (purple) in CSE-treated neutrophils (n = 3). Blue, DAPI-stained nuclei. Scale bar = 2 µm. e Real-time Ca2+ flux dynamics in neutrophils with or without KB-R7943 administration measured by non-invasive micro-test technique (n = 6). Upward bars represent Ca2+ efflux activity, and downward bars represent Ca2+ influx. f, g Simultaneous real-time measurements of intracellular Ca2+ (F340/380) and Na⁺ (F500/545) in neutrophils by fluorescent probes upon stimulation with CSE, PMA, or fMLP (n = 3). h Ca2+ influx in bone marrow-derived neutrophils from Slc8a1f+/f+;Mrp8-Cre- and Slc8a1f+/f+;Mrp8-Cre+ mice upon CSE stimulation (n = 3). i ELIZA detects Cit-H3 expression in the cell culture of CSE-stimulated bone marrow-derived neutrophils (n = 6). j NETs formation in CSE-stimulated neutrophils is visualized by scanning electron microscope (n = 3). Scale bar = 2 µm. k Effects of KB-R7943, BAPTA-AM, and A-23187 on NETs area in CSE-stimulated neutrophils. Scale bar = 2 µm. l ELIZA detects Cit-H3 expression in unstimulated neutrophils and CSE-stimulated neutrophils with KB-R7943, BAPTA-AM, and A-23187 treatment (n = 5). Each data point represents one biologically independent replicate with 3 technical replicates (a, b, i, l). Data shown in Fig. 7e represent the mean of measurements (6 biological replicates). Data in Fig. 7f–h represent the mean of measurements (3 biological replicates with 3 technical replicates). All quantitative data are presented as Mean ± SD. Two-sided t-test (i) and one-way ANOVA with Tukey’s multiple comparison test (a, b, l) 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. CSE cigarette smoke extract; PMA Phorbol 12-myristate 13-acetate; fMLP N-Formylmethionyl-leucyl-phenylalanine; LPS lipopolysaccharide; Cit-H3 Citrullinated Histone H3; WT wild type; min minutes; ns no significance.

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