Fig 2: 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 3: 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 4: Growth of Rhodococcus ruber R1 on several lignocellulose-related aromatic compounds. The growth of strain R1 on 2 mM guaiacol (A), p-cresol (B), p-coumarate (C), phenol (D), m-cresol (E), and 4-hydroxybenzoate (F) as the sole carbon and energy sources was assessed. All reagents were of analytical grade and were obtained from Sigma-Aldrich (Buchs, Switzerland). The cultures were incubated in a 96-well microplate (Thermo Fisher Scientific, Rochester, NY, USA), with agitation, for 15 min at 30°C, and the optical density at 600 nm (OD600) was measured with a Synergy HTX multimode reader (BioTek, Winooski, VT, USA). Minimal salt medium without carbon sources (control) supported minor growth, which was subtracted from the growth curves for medium with carbon sources. Cell cultures were inoculated with 100-fold dilutions of overnight cultures of strain R1 grown on R2A broth. Five replicates were performed for growth measurements. Error bars represent 1 standard deviation.

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).