Fig 2: Reactive oxygen species scavenger inhibits the production of oxidized mitochondrial DNA and NETs. Bone marrow neutrophils were stimulated with mitochondrial DNA (5 µg/mL) for 2 h. Sytox Green (5 µmol/L; Life Technologies) was used to detect extracellular DNA. Fluorescence intensity was quantified using the BIOTEK plate reader Synergy HTX, n = 8 (A). Neutrophils were stimulated with mitochondrial DNA (5 µg/mL) for 30 minutes, and ROS production was determined, n = 7 (B). Neutrophils were stimulated with mtDNA (5 µg/mL) or in the presence of 5 mmol/L NAC for 30 minutes, and oxidized mitochondrial DNA was detected with biotinylated TOMM20 and 8-OHdG antibodies, n = 7 (C). Primary lung cells were stimulated with bleomycin (20 µg/mL) or in the presence of 5 mmol/L NAC for 24 h. TOMM20 and 8-OHdG fluorescence microscopy (D) or by flow cytometry was performed, n = 8 (E). Freshly isolated neutrophils were cultured with mitochondrial DNA or oxidized mitochondrial DNA (0.5-10 µg/mL) for 2 h. Fluorescence intensity of Sytox Green was quantified using the BIOTEK plate reader Synergy HTX, n = 8. Black for mitochondrial DNA and red for oxidized mitochondrial DNA (F). Neutrophils were cultured with mitochondrial DNA or oxidized mitochondrial DNA (5 µg/mL) for 2 h. Western blots for detecting the proteins PAD4 and Rac2 were performed (G). Data are representative of three independent experiments, and the results are expressed as the means ± SEM. Statistical comparisons were performed using Student's t test or Dunnett's t test (*P < 0.05; **P < 0.01; ***P < 0.005)

Fig 3: Mitochondrial DNA induces neutrophil extracellular trap formation. Bone marrow neutrophils from C57BL/6 mice were obtained and stimulated with mitochondrial DNA (5 µg/mL) and mitochondria (100 µg/mL) for 2 h. Cells were stained with an anti-histone H3 primary antibody and the appropriate second antibody. DAPI was used to stain DNA. Images were acquired using a confocal microscope (Zeiss LSM 510 Meta) (A). Neutrophils were cultured (1 × 106 cells/well) and stimulated with fMLF (1 µmol/L), mtDNA (5 µg/mL) or mitochondria (100 µg/mL) for 2 h in 6-well plate at 37°C. The elastase production in the supernatant was measured by ELISA, n = 6 (B). Sytox Green (5 µmol/L; Life Technologies) was used to detect extracellular DNA. Fluorescence intensity was quantified using the BIOTEK plate reader Synergy HTX, n = 6 (C). Neutrophils were stimulated for 2 h with 5 µg/mL mtDNA, 1 µg/mL LPS and 25 nmol/L DNaseI. The change in neutrophil morphology was observed by Giemsa staining (D). The freshly isolated neutrophils were cultured with mitochondrial DNA (5 µg/mL) at a concentration of 2 × 106cell/ml for 2 hours. Western blot analysis was performed (e). Blot intensities were analysed by Image J (F-I). Data are representative of three independent experiments, and the results are expressed as the means ± SEM. Statistical comparisons were performed using Student's t test or Dunnett's t test (*P < 0.05; **P < 0.01; ***P < 0.005)

Fig 4: Paracoccus aminovorans (Pa) increases biofilm production in Vibrio cholerae (Vc).Crystal violet assays were performed in 96-well microtiter plates to quantify biofilm formation. Overnight-grown (A) wild-type Vc or (B) ?vpsL mutant and Pa cultures were diluted to a final concentration of 106 colony-forming unit (CFU) in a total volume of 200 µL/well. In samples containing a 1:10 ratio of Vc/Pa, Pa was diluted to a final concentration of 107 CFU. Samples with heat-killed (HK) Pa are specified on the x-axis. Microtiter plates were incubated at 37°C for 24 hr. Crystal violet staining and ethanol solubilization were performed as previously described (O’Toole, 2011). Absorbance of the crystal violet stain was measured at 550 nm using a BioTek Synergy HTX plate reader. Data are represented with horizontal lines indicating the mean with standard deviation. Unpaired U-test (Mann–Whitney); **p=0.01. (C) Vc and Pa were co-cultured at a 1:10 ratio statically for 72 hr at 30°C. At the specified time points, culture from the air–liquid interface was sampled and the RNA extracted. Relative vpsL transcript levels were determined by qRT-PCR using housekeeping gene groEL and the ??CT analytic method. Bars on graphs depict mean with standard error of mean (SEM). Mann–Whitney U-test was performed, **p<0.01, *p<0.05, NS p>0.05. Figure 5—source data 1.Crystal violet assays of biofilm formed by Vc mono- and Vc/Pa co-cultures using WT with live or heat-killed Pa. Figure 5—source data 2.Crystal violet staining of biofilm formed by Vc mono- and Vc/Pa co-cultures using vpsL mutants with live and heat-killed Pa. Figure 5—source data 3.qRT-PCR analysis of vpsL gene in cells in biofilm formed by Vc mono- and Vc/Pa co-cultures.

Fig 5: Porcine a-tocopherol concentrations in serum do not increase until the dose is higher than 100 mg/kg. The relative concentrations of a-tocopherol in the samples from weaned pig's serum were detected in microplate reader (BioTek, SYNERGY HTX, VT, USA) at 490 nm by the a-tocopherol enzyme-linked immunosorbent assay (ELISA) kit. All data were represented as mean ± SD (n = 3, a < 0.05 vs. control).