U75302

J774.1 macrophages were plated at the density of 2 × 10⁵ cells per well in 200 μl of serum-free RPMI supplemented with antibiotics. The cells were then cultured at 37 °C in 5% CO₂ for 2 h. Next, the supernatants were removed, and the cells were treated or not with specific inhibitors/antagonists for 30 min: indomethacin (10 μM; Cayman Chemical); AH6809 (1 μM; Cayman Chemical); AH23848 (1 μM; Cayman Chemical); U-75302 (0.1 and 1 μM; Cayman Chemical); and NFκB Activation Inhibitor (20 nM; Calbiochem, Darmstadt, Germany). H89 dihydrochloride hydrate (25 μM; Sigma-Aldrich) was added for 2 h in the cell culture medium before stimulation. AH6809 and U-75302 from ethanol stock solutions were diluted in cell culture medium and the same concentration of ethanol (maximum 0.1%) was added to the medium only (control). The AH23848 and NFκB inhibitor from DMSO stock solutions were diluted in the cell culture medium and the same concentration of DMSO (maximum 0.1%) was added to the medium only (control). All compounds were diluted in 200 μl of serum-free DMEM, and the same solution with solvent diluents was used as control. After treatment, the cells were stimulated with TsV (50 μg ml⁻¹) under the same experimental conditions and after 24 h at 37 °C in a humidified atmosphere 5% of CO₂, the supernatants were collected for IL-1β quantification.

Resident peritoneal macrophages and bone marrow-derived neutrophils (2 × 10⁵/well) were plated into two 96-well plates with opaque walls and clear bottoms. Cells were cultured in DMEM and pretreated with 10 μM U75302 (Cayman Chemicals, Ann Arbor, MI) for 30 minutes or 10 nM LTB₄ for 5 minutes before the addition of MRSA-GFP at a multiplicity of infection of 50:1, as we have previously described [49 (link)]. Infected cells were incubated 1 hour to allow phagocytosis, and both plates were washed with warm PBS. The PBS and treated samples were added to the killing plate and incubated for another 2 hours for killing assays. To measure the intensity of GFP fluorescence, extracellular fluorescence was quenched with 50 μL of 500 μg/mL trypan blue, and the GFP fluorescence was quantified using a fluorimeter plate reader. Trypan blue served as blank. A reduction in GFP fluorescence in the killing plate relative to the phagocytosis plate indicated bacterial killing.

Female C57BL/6 (B6) mice were purchased from Japan SLC (Hamamatsu, Japan). All experiments were conducted on 8- to 12-week-old mice. Kaede transgenic mice and BLT1-deficient mice on a B6 background were generated as previously described^{20 (link),35 (link)}. RvE1 was synthesized as previously described^{36 (link)}, and was stored in −80 °C at the concentration of 1 mg/ml in ethanol, and was prepared at the indicated concentration every time for each experiment. LTB4 and a BLT1 antagonist (U-75302) were purchased from Cayman Chemical (Ann Arbor, MI).

Zileuton and alpha-smooth muscle actin (α-SMA) antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). LTB4, U75302 and REV5901 were from Cayman Chemical Inc (Ann Arbor, MI, USA). MK 886 was purchased from EMD Serono (Rockland, MA, USA), HMGB1 from R&D systems (Minneapolis, MN, USA), CD11b antibody from Abcam (Cambridge, MA, USA), and BLTR1 antibody from Biorbyt (Cambridge, UK). CD36, CD14, β-actin, and 5-LO antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), R-phycoerythrin PE-conjugated mouse anti-human CD11b/Mac-1 antibody and PE-conjugated mouse IgG isotype control antibody from BD (San Diego, CA, USA). Horseradish peroxidase (HRP)-conjugated IgG antibody was used as the secondary antibody from Santa Cruz Biotechnology. Restriction enzymes were purchased from Promega (Madison, WI, USA). 5-LO and BLTR1 siRNA oligonucleotides were synthesized by Bioneer (Daejeon, ROK). siRNA molecules were transfected into cells using Lipofectamine 2000 siRNA transfection reagent (Invitrogen, Carlsbad, CA, USA). PCR primers were from Bioneer.