Docosahexaenoic acid

Cells were seeded at 9 × 10⁴ in a 12-well plate. After 24 h, the cells were treated with 100 µM gamma-linolenic acid, eicosapentaenoic acid, or docosahexaenoic acid (Cayman Chemical, Ann Arbor, MI, USA) complexed with fatty-acid-free bovine serum albumin (ALB) (Sigma Aldrich, Brazil) as previously described [37 ]. ALB was used as the vehicle control (Sigma Aldrich, Brazil). Cells were concomitantly grown in the presence or absence of 25 µM TMZ. Culture medium and treatments were changed every 24 h. After 72 h of treatment the cells were trypsinized, followed by centrifugation at 250× g for 5 min. The cell pellet was rinsed with ice-cold PBS, centrifuged, and resuspended in ice-cold 70% ethanol for at least 24 h. After ethanol fixation, cells were again washed with ice-cold PBS and incubated for 30 min with 500 μL of a staining solution (20 μg/μL propidium iodide, 50 μg/μL RNAse A and 0.1% Triton X-100). At the end of incubation, cells were centrifuged, resuspended in ice-cold PBS, and kept on ice before analysis. Cell cycle phase was determined by propidium iodide fluorescence detection of 10,000 events in a Guava^® easyCyte flow cytometer (Millipore, Burlington, MA, USA) using a λ_ext. of 532 nm for propidium iodide detection [41 (link),42 (link)].

Klebsiella pneumoniae ATCC13883 was used in this study. CM9 and G56 minimal media (0.4% Glucose, 0.4% casamino acids (Fisher BioReagents), supplemented with 150 mmol/L NaCl) were used for growth of bacteria in experiments, except for sole carbon source experiments which were performed in M9 minimal media lacking glucose. All experiments were performed at 37°C. Fatty acids used in this study were purchased from Cayman Chemicals [linoleic acid (18:2), α‐linolenic acid (18:3α), γ‐linolenic acid (18:3γ), dihomo‐γ‐linolenic acid (20:3), arachidonic acid (20:4), eicosapentaenoic acid (20:5), and docosahexaenoic acid (22:6)] and administered at a concentration of 300 μmol/L for each experiment, except for sole carbon source where they were administered at 1 mmol/L.

Cells were grown in culture medium as detailed in Section 2.1, in 25 cm² flasks in the presence of 100 µM gamma-linolenic acid, eicosapentaenoic acid, or docosahexaenoic acid (Cayman Chemical, Ann Arbor, MI, USA) complexed with fatty-acid-free bovine serum albumin (ALB) (Sigma Aldrich, São Paulo, Brazil) as previously described [37 ]. ALB was used as the vehicle control (Sigma Aldrich, São Paulo, Brazil). Cells were concomitantly grown in the presence or absence of 25μM TMZ. Culture medium and treatments were changed every 24 h.
After 72 h, cells were collected by trypsinization, centrifuged at 250× g for 5 min, washed with PBS, and the pellets resuspended in DMEM containing rhodamine 123 (R123) (Cayman Chemical, Ann Arbor, MI, USA). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂ for 60 min, then centrifuged and washed with PBS. The cells were placed in fresh DMEM and incubated at 37 °C in a humidified atmosphere with 5% CO₂. At 20 min intervals aliquots were collected and cells were separated from the medium by centrifugation, washed with PBS, and resuspended in PBS. The medium and the resuspended cells were placed separately in a 96-well plate and fluorescence was determined at λ_ext. 490 nm and λ_em. 530 nm using a Synergy H1 Hybrid Multi-mode Reader (Biotek, Winooski, VT, USA).

LC-MS/MS-based lipid mediator and oxylipin profiling were carried out as described elsewhere [4 (link)]. A QTrap 6500 mass spectrometer (Sciex) was used, coupled to a Shimadzu Nexera LC30-system including auto-sampler and column oven (Shimadzu). The column was a Kinetex C18 50 × 2.1 mm, 1.7 μm, protected with a C8 pre-column (Phenomenex). LC-MS/MS peaks were integrated with manual supervision, and the areas were corrected to corresponding IS using MultiQuant™ 2.1 (Sciex). For quantitation, the multiple reaction monitoring (MRM) transitions and collision energies (CE) were used together with calibration lines for quantification. Calibration lines were constructed using 15-HETE, 17R-HDHA, leukotriene B₄, prostaglandin E₂, arachidonic acid, docosahexaenoic acid, and eicosapentaenoic (Cayman Chemicals)

Docosahexaenoic acid, and diclofenac were obtained from Cayman Chemical (Ann Arbor, MI, USA). Indomethacin, naproxen, piroxicam, resazurin, horseradish peroxidase-labeled mouse anti-β-actin, anti-K-Ras, anti-N-Ras, and anti-H-Ras antibodies were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Antibodies specific for Bax, Bim, Mcl-1, Bcl-xL, PARP, cleaved PARP, pro-caspase 3, pro-caspase 7, cleaved-caspase 3, phospho-MEK1/2-Ser217/221, phospho-p44/42 MAPK (Erk1/2)-Thr202/Tyr204, phospho-p90RSK-Ser380, phospho-Akt (Ser473), Akt (pan), horseradish peroxidase-labeled anti-mouse, and anti-rabbit immunoglobulins were obtained from Cell Signaling Technology (Beverly, MA, USA). The Ras Activation Assay Kit was purchased from Cytoskeleton Inc. (Denver, CO, USA).

The culture media RPMI 1640, HEPES, gentamycin, and Albumax I were obtained from GIBCO Life Technologies (Thermo Fisher Scientific, Waltham, MA, USA). Glucose, sodium bicarbonate, and hypoxanthine were purchased from Millipore-Sigma (Burlington, MA, USA). Polyprenol, dolichol, polyprenal, and nor-dolichol mixtures of 13 to 21 isoprene units were obtained from Avanti Polar Lipids (Alabaster, AL, USA). The dolichyl phosphate mixture (14 to 18 isoprene units), all trans-polyprenol (9 isoprene units), and isopentenol were obtained from Isoprenoids LC (Tampa, FL, USA). The following chemicals were obtained from Cayman Chemicals (Ann Arbor, MI, USA): polyunsaturated fatty acid mixture (adrenic acid, arachidonic acid, dihomo-γ-linolenic acid, docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, linoleic acid, α-linolenic acid, and γ-linolenic acid, stearidonic acid), saturated and monounsaturated fatty acid mixture (arachidic acid, lauric acid, lignoceric acid, myristic acid, nervonic acid, oleic acid, palmitic acid, palmitoleic acid, and stearic acid), and ubiquinone-10. Squalene, 2,3-oxidosqualene, cholesterol, β-carotene, menaquinone-4, α-tocopherol, phylloquinone (vitamin K1), retinal, retinoic acid, and retinol were purchase from Millipore-Sigma (St. Louis, MO, USA). Anhydrotetracycline was obtained from Cayman Chemical (Ann Arbor, MI, USA).