Deferoxamine mesylate

Raw 264.7 murine macrophages were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in tissue culture flasks. Raw cells were harvested via trypsinization and subcultured at 5 × 10⁵ cells per well in 24-well plates for 24 h to allow attachment overnight before experimentation.
For BMM preparation, femurs and tibias were removed from euthanized female CD1 mice and flushed with sterile PBS through a 70 µm filter. The cells were centrifuged for 5 min at 4 °C and 500× g and resuspended in 28 mL complete DMEM (cDMEM) (10% FBS, 5% horse serum, 1% HEPES, 1% sodium pyruvate, 1% GlutaMAX, 1% penicillin–streptomycin). Then, L929 conditioned media (7 mL) containing murine CSF was prepared and added to the cDMEM, as described previously [21 (link)]. Macrophage differentiation was carried out for 12 days before experimentation.
Macrophages (Raw 264.7 or BMMs) were then treated with purified LT, purified ST, Salmonella minnesota LPS (InvivoGen, cat# R595, San Diego, CA, USA), IL-33 (R&D Systems, cat# 3626-ML, Minneapolis, MN, USA), forskolin (Sigma, cat#F3917, Waltham, MA, USA), CFA/I (BEI Repository, cat# NR-49110, Manassas, VA, USA), or deferoxamine mesylate (Sigma, cat# D9533).

Nuclease P1 from Penicillium citrinum, phosphodieasterase I and II, alkaline phosphatase from bovine intestinal mucosa, DNase I and DNase II, benzonase 99%, BHT, deferoxamine mesylate and pentostatin were purchased from Sigma-Aldrich (Steinheim, Germany). RNase T1 was from Thermo Fisher Scientific (Waltham, MA, USA) and RNase A from Roche Diagnostic GmbH, (Mannheim, Germany). The 3 kDa cut-off filters were obtained from Millipore (Bedford, OH, USA). Solvents (HPLC-grade) were purchased from Fisher Scientific. 2′-Deoxyadenosine monohydrate were purchased from Berry & Associates Inc. (Dexter, NY, USA). Isotopic labeled internal standards of 5′R-cdA, 5′S-cdA, 5′R-cdG, 5′S-cdG and 8-oxo-dA (see Supplementary Materials) were prepared according to the previously reported procedures [21 (link)]. Ultrapure water (18.3 MΩ.cm) distilled and deionized water (Milli-Q water) were purified by a Milli-Q system (Merck-Millipore, Bedford, OH, USA). Chloroform, methanol and n-hexane were purchased from Merck (HPLC-grade). Anhydrous sodium sulfate (Na₂SO₄) was purchased from Carlo Erba (Val de Reuil Cedex, France). All fatty acid methyl esters (FAME) used as reference standard for GC analyses were purchased from Sigma-Aldrich or Fluka (Steinheim, Germany) without further purification. Analytical silica gel thin-layer chromatography was performed on Merck silica gel 60 plates.

Nuclease P1 from Penicillium citrinum, phosphodieasterase I and II, alkaline phosphatase from bovine intestinal mucosa, DNase I and DNase II, benzonase 99%, BHT, deferoxamine mesylate and pentostatin were purchased from Sigma-Aldrich (Steinheim, Germany). RNase T1 was from Thermo Fisher Scientific (Waltham, MA, USA) and RNase A from Roche Diagnostic GmbH, (Mannheim, Germany). The 3 kDa cut-off filters were obtained from Millipore (Bedford, OH, USA). Chemicals for the synthesis of oligonucleotides were purchased from Sigma Aldrich, Fluka and Link Technologies. CuCl₂, L-methionine, L-ascorbic acid and alkaline phosphatase were purchased from Sigma-Aldrich. Hydrogen peroxide (30%) and solvents (HPLC-grade) were purchased from Fisher Scientific. 2′-deoxyadenosine monohydrate and 2′-deoxyaguanosine were purchased from Berry & Associates Inc. (Dexter, USA). Isotopic labeled internal standards of 5′R-cdA, 5′S-cdA, 5′R-cdG, 5′S-cdG, 8-oxo-dG and 8-oxo-dA (see Supporting Information) were prepared according to the previously reported procedures [31 (link)]. Ultrapure water (18.3 MΩcm) distilled and deionized water (Milli-Q water) were purified by a Milli-Q system (Merck-Millipore, Bedford, OH, USA).

Synechococcus sp. PCC7002 was cultivated in the laboratory with Medium, at 30 °C, under a light intensity of 140 μmol m⁻² s⁻¹, in 10 L glass photobioreactors (Figure 5). All Wistar rats were obtained from Shandong Lukang Pharmaceutical Group Co., Ltd. (Shandong, China). Pelletized purified AIN-93G-based diets were obtained from TROPHIC Animal Feed High-Tech Co., Ltd. (Nantong, China). Calcein acetoxymethylester (calcein-AM), cell culture medium (DMEM), and fetal bovine serum were purchased from Gibco (Grand Island, NY, USA). XAD-2 resin, deferoxamine mesylate, sodium phytate, and bathophenanthrolinedisulfonic acid (BPDS) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). All other reagents were analytical grade or better.

ROS are formed in mitochondria and to high extent also in other cellular compartments of lung samples [24 (link)]. Therefore, total cytoplasmic protein was isolated from lung tissue in ice-cold 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (5 μl∙mg^− 1 wet tissue) containing 1 μg∙ml^− 1 saponine (Sigma, Saint Louis, MO) as mild detergent and 50 μM deferoxamine mesylate (Sigma) as Fe³⁺ chelator. Nuclei were then removed by centrifugation (600 g for 10 min). 10 μl nuclei-free cytoplasmic fraction were mixed after 90 min (Fe³⁺ chelate effect completed) with 90 μl 1 mM 1-hydroxy-3-carboxy-2,2,5,5,-tetramethyl-pyrrolidine (CPH; Noxygen, Elzach, Germany) as a spin trap for O₂^•-. A computer-operated electron paramagnetic resonance spectrophotometer (MiniScope MS100; Magnettech, Berlin, Germany) equipped with MiniScope Control v2.7.3 analysis software (Microtech GmbH, Bad Kreuznach, Germany) measured the time-dependent formation of 3-carboxy-2,2,5,5-tetra-methyl-pyrrolin-1-oxyl (^•CP) at room temperature every 10 min. O₂^•- data were expressed as ^•CP formation rate∙min^− 1.

The 8-oxodGuo (>98% purity), 2-deoxyguanosine (dGuo; >98% purity), guanosine (Guo; 98% purity), deferoxamine mesylate (DFOM), and HPLC-grade methanol were obtained from Sigma-Aldrich Inc., USA. 8-oxoGuo (>98% purity) was obtained from Alexis Biochemicals (San Diego, CA, USA). HPLC-grade ammonium acetate was obtained from Fisher Scientific, USA. Heavy-isotope-labeled 8-oxo-[¹⁵N₅]dGuo, [¹⁵N₅]dGuo, and [¹⁵N₅]Guo were obtained from Cambridge Isotope Laboratories (Andover, MA, USA), and 8-oxo-[¹⁵N₂¹³C₁]Guo was obtained from Toronto Research Chemicals (Toronto, Canada). Water was deionized at 18.2 MΩ.