Mk886

The mouse kidney tubular cell line MM55.K (ATCC ref. CRL-6436) was maintained in DMEM (Gibco, #11965092) supplemented with 10% fetal bovine serum (FBS), at 37 °C, 5% CO₂ atmosphere and split with trypsin/EDTA solution. Cells were plated onto 6-well plates (3 × 10⁵ cells/well) and, after 24 h of culture, treated with gemfibrozil (100 µM), MK-886 (25 µM, Cayman Chemical, #10133), a PPARα antagonist, and/or metformin (50 µM) in DMEM containing 10% FBS. After 24 h in culture, cells were briefly washed with PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄). RNA extraction, cDNA synthesis, and Real-Time PCR were then conducted.

Two osteotropic metastatic human breast cancer cell lines were used in this study: MDA-MB-231/B02 (MDA-B02) and a subclone expressing the three markers GFP, luciferase, and a beta-galactosidase referred to as MDA-B02-Luc for simplification and which was described previously [46 (link)], as well as their parental breast cancer cell line MDA-MB-231 (MDA-231) and the murine 4T1 breast cancer cells obtained from the American Type Culture Collection (ATCC^®). The 4T1 cells derive from a BALB/c spontaneous mammary carcinoma and are naturally resistant to 6-thioguanine [47 (link)]. They were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Zafirlukast and montelukast, the CysLT1R antagonists, everolimus, the mTORC1 inhibitor, and clopidogrel, the P2Y₁₂ inhibitor, were purchased from Sigma. HAMI3379, the CysLT2R antagonist, and MK-886, the FLAP inhibitor, were from Cayman. LTD₄ was from Bertin.

PBMCs were isolated using Ficoll-Paque PLUS (GE Healthcare, Uppsala Sweden) density gradient centrifugation following the manufacturer’s instructions. PBMCs were plated at the density of 1 × 10⁶ cells/well in 300 µL serum-free RPMI supplemented with antibiotics (incomplete RPMI-1640). The adhered cells were then cultured at 37°C (5% CO₂) for 2 h. Next, the cells were pre-incubated with or without indomethacin (30 min; 10 µM; Cayman Chemical, MI, USA), MK886 (30 min; 10 µM), PGE₂ (10 min; 10 µM; Cayman Chemical), LTB₄ (10 min; 100 nM; Cayman Chemical), or dexamethasone (1 h; 0.1 µM; Aché Laboratórios, São Paulo, Brazil). PGE₂ and LTB₄ from ethanol stock solution were suspended in serum-free RPMI, and an equivalent volume of ethanol (not more than 0.01%) was added to the RPMI; this solution was designated as the vehicle control. indomethacin, MK886 (prediluted in PBS/0.01% ethanol), and dexamethasone were diluted in serum-free RPMI. RPMI containing the same proportion of ethanol was used to dilute the compounds, yielding the vehicle control. After treatment, PBMCs were stimulated with TsV (50 µg/mL) for 24 h at 37°C in a humidified atmosphere with 5% CO₂. Subsequently, the supernatants were collected for IL-1β quantification, and the cell lysate was used for analysis of gene expression.

Human umbilical vein endothelial cells (HUVEC) were purchased from ATCC (PCS-100-013) and Lonza Biosciences (Basel, CH, pooled donors, catalog no. C2519A). Culture media and supplements (EBM-2™ medium and EGM-2 Bullet kit) were purchased from Lonza (Basel, CH). Trypsin-EDTA solution (0.25%-0.9 mM) was purchased from ThermoFisher Scientific (Waltham, MA). Cytokines (human recombinant interleukin−1β, IL−1β and tumor necrosis factor−α, TNFα) were purchased from R&D Systems (Minneapolis, MN). Primary antibodies were supplied by Abcam (Waltham, MA), Invitrogen/ThermoFisher Scientific (Waltham, MA), Sigma/Aldrich (St. Louis, MO), ProteinTech (Rosemount, IL) and Cayman Chemical Co. (Ann Arbor, MI) (S1 Table). Secondary antibodies (goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP) were purchased from Invitrogen/ThermoFisher Scientific. Enzymes, buffers and fetal bovine serum were purchased from GIBCO (ThermoFisher). Aspirin and salicylic acid were obtained from Sigma/Aldrich. Arachidonic acid, nordihydroguariatic acid (NDGA), Zileuton and MK-886 were purchased from Cayman Chemical Co. (Ann Arbor, MI). All other chemicals were tissue culture or reagent grade Sigma-Aldrich.

MK-886 (Cayman), an inhibitor of the 5-LO activating protein (FLAP) was injected systemically (3–10 mg/kg, i.p.) and intracerebroventricularly (250–500 pmol/2 µl, i.c.v.). This drug had already been tested in our previous paper, and a dose of 3 mg/kg was sufficient to inhibit half of AEA-induced catalepsy [1] (link). Doses ranging from 3 to 10 mg/kg also influence mouse behavior in the forced swim test [8] (link). The direct 5-LO inhibitor zileuton (Cayman) was given orally in a selected high dose (40 mg/kg, p.o.), which is roughly 25% above the dose known to inhibit 50% of the enzyme activity (ED₅₀) in the periphery [9] (link). Both drugs were injected 1 h before behavioral experiments and 10% DMSO was used as vehicle. AM251 (1–3 mg/kg) and lipoxin A₄ (1–10 µg) were injected i.p. 30 min before testing.

To investigate the effect of LCMs on LTC₄S activity, we used microsomal membranes from HEK-293 cells stably expressing LTC₄S^{70 (link)}. Membranes (2.5 µg total protein) were treated with LCMs for 10 min at 4 °C in potassium phosphate buffer (0.1 M, pH 7.4) containing 5 mM glutathione. The conversion of LTA₄ to LTC₄ methyl esters was initiated by addition of LTA₄ methyl ester (Cayman Chemicals; 1 µM). After 10 min at 4 °C, the reaction was terminated by ice-cold methanol (one volume), and LTC-d5 methyl ester (5 ng; Cayman Chemicals) was added as internal standard. LTC₄ was extracted and analyzed by UPLC-MS/MS as described for lipid mediators. Vehicle (DMSO)-treated microsomal membranes produced 7.1 ± 0.5 nmol LTC₄. The LTC₄ reference inhibitor MK-886 (Cayman Chemicals, 10 µM) suppressed LTC₄ formation by 95.6 ± 0.9%.

Mouse monoclonal antibody for α-tubulin, C23, NF-κB p65 and rabbit polyclonal antibody for IgG, IKKα/β, IκBα, NF-κB p50, NF-κB p65 and goat anti-mouse or anti-rabbit secondary antibody conjugated with horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal antibody for phosphor-IκBα and rabbit monoclonal antibody for phosphor-IKKα/β were from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-5-LOX antibody was from Novus (Littleton, CO, USA). 5-LOX inhibitors, including MK-886, Nordihydroguaiaretic acid (NDGA); leukotriene B4 and leukotriene B4 receptor antagonist LY29311 were from Cayman Chemical Company (Ann Arbor, MI, USA). Collagenase and 4′,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human TNF-α and enhanced chemiluminescent HRP substrate (ECL) were from Millipore (Bedford, MA, USA). We purchased RPMI-1640 medium, trypsin and anti-rabbit secondary antibody conjugated with Alexa Fluor 488 from Invitrogen (Carlsbad, CA, USA) and fetal bovine serum (FBS) from Biological Industries (Kibbutz Beit Haemek, Israel). Tri-zol was from MDBIO (Taipei, Taiwan). MMLV Reverse Transcriptase kit was from Promega (Madison, WI, USA). Taqman PCR Master Mix and qPCR probes were from Applied Biosystems/Invitrogen (Foster city, CA, USA).