Fig. 2.

Cigarette smoke extract (CSE)-induced NETosis requires mitochondrial reactive oxygen species (ROS) and mitochondrial respiratory chain, but not nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Statistical analysis: n = 3–10 for each bar in (a–f, h, i, k), from at least three healthy participants or three patients with COPD, data were presented as the mean ± standard deviation; Differences are assessed by the (a–f, k) one-way or (h, i) two-way ANOVA analysis of variance, followed Tukey’s honest significant test; P < 0.05 represents a significant difference, the scattered samples and the p values are displayed in figures. a–f The effects of several chemicals on the percentage of NETosis of human peripheral blood neutrophils (hPBNs, derived from healthy participants), stimulated by 5% CSE and 50 nM of PMA for 18 h: a 50 μM of mitoTEMPO (a mitochondrially targeted antioxidant), b 50 μM of thenoyltrifluoroacetone (TTFA, a mitochondrial respiration inhibitor), c 50 μM of diphenyleneiodonium chloride (DPI, a NADPH oxidase inhibitor), d 50 μM of VAS2870 (VAS, a NADPH oxidase inhibitor), e 200 IU/mL of deoxyribonuclease-I (DNase-I, an endonuclease for single- and double-stranded DNA) and f 50 μM of GW311616A (a selective human neutrophil elastase inhibitor), as characterized by immunofluorescence staining of NETs components (supplementary Method 7). g–i The incubation of 5% CSE for 2 or 4 h induces the release of mitochondrial ROS (stained by mitoSOX Red, supplementary Method 8, 17) by hPBNs from both healthy participants and patients with COPD, as assessed by i flow cytometry and fluorescence staining (scale bar: 50 μm), and summarised in g the percentage of positive events and h the mean intensity of mitoSOX Red. j The immunofluorescence colocalization of 8-hydroxy-2’-deoxyguanosine (8-OHdG, a marker of oxidative stress to DNA, green) and translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20, a mitochondrial outer membrane marker, red), with and without the treatment of 5% CSE (supplementary Method 8). Note: oxidative-damaged DNA are presented on the mitochondrial membrane following the treatment of 5% CSE for 2 h (scale bar: 10 μm). k The fluorescence intensity (mean) of 8-OHdZG on hPBNs treated with 5% CSE and 50% CSE from healthy participants and patients with COPD