Gw1929

Cells of murine macrophage RAW264.7 (Cell Bank of the Chinese Academic of Sciences) were cultured in DMEM solution containing 10% FBS with 100 U/mL penicillin G and 100 U/mL streptomycin sulfate at 37 °C with 5% CO2. All the experiment intervention was conducted on the third passage of cells. RAW264.7 macrophages were incubated with either PA (0.5 mmol/L) or DHA (50 μmol/L) alone for 6 h or 24 h, with DMEM solution and LPS (100 ng/mL) or IL-4 (5 ng/mL) treatment as control respectively. For PPAR-γ agonist or antagonist intervention, RAW264.7 macrophages were pre-incubated with either GW1929 (an agonist of PPAR-γ, 20 μmol/L, Sigma Aldrich) or GW9662 (an antagonist of PPAR-γ, 60 μmol/L, Sigma Aldrich) for 3 h, followed by combined treatment of different fatty acids with PPAR-γ agonist or antagonist as above.

C3H10T1/2 and 3T3-L1 cells were purchased from the American Type Culture Collection (Rockville, MD, USA) and were maintained as previously described [42 (link)]. 3T3-L1 cells were induced into adipocytes in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, Logan, UT, USA) media supplemented with 10% FBS, 1 μM dexamethasone (Sigma, St. Louis, MO, USA), 0.5 mM isobutyl-1-methylxanthine (Sigma, St. Louis, MO, USA) and 5 μg/mL insulin (Sigma). After 48 h, the differentiating cells were refreshed with media containing DMEM, 10% FBS and 5 μg/mL insulin. C3H10T1/2 cells were further supplemented with 20 nM GW1929 (Sigma) to induce adipocyte differentiation. T37i cell lines, a brown preadipocytes, kindly provided by M. Lombes (Paris-Sud University, Le kremlin Bicêtre, France) were maintained DMEM/F-12, 10% CS, and 1% penicillin/streptomycin. Confluent cells were incubated in DMEM/F-12, 10% CS, 5-μg/ml insulin and 2.5 nM T3, to induce brown adipocytes. After adipocyte differentiation for 6–8 days, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and stained with 0.5% Oil Red O (Sigma).

HepG2 and Huh-7 cells were obtained from the American Type Culture Collection (ATCC, U.S.A.) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (ThermoFisher Scientific, U.S.A.) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, U.S.A.) at 37°C and 5% CO₂. For NAFLD cellular model, HepG2 and Huh-7 cells were assigned into control and free fatty acid (FFA; mixture of 1 mM FFAs, containing oleic acid and palmitic acid at 2:1 volume ratio) treatment group to induce steatosis of different degree, and were used for further assays. In the latter group, cells were treated with concentrations of 1 mM FFA for 24 h, and were used for further assays. The PPARγ agonist GW1929 and the PPARγ antagonist GW9662 were purchased from Sigma–Aldrich. HepG2 and Huh-7 cells were treated with 20 μM of GW1929 and 10 μM of GW9662 for 1 h, followed by FFA treatment.

Dihydrotestosterone was purchased from Steraloids. Vitamin K antagonists were purchased from: warfarin sodium (TCI Chemicals), scopoletin (Sigma), brodifacoum (Sigma). Bicalutamide was purchased from Sigma. PPAR agonists and antagonists were purchased from: GW1929 (Sigma), Pioglitazone (Santa Cruz), GW9662 (Sigma).

FL-DCs were prepared as previously described (37 (link)), with some modifications. In brief, bone marrow cells were cultured at a concentration of 3×10⁵ cells/ml in RPMI-1640 medium containing 10% FBS supplemented with 200 ng/ml recombinant murine Flt3L (rmFlt3L, PeproTech, Rocky Hill, NJ, USA) and 2-mercaptoethanol (50 μM, Sigma-Aldrich), for 8 d. Cells were treated with MEHP, GW9662 (PPARγ antagonist; Tocris, Bristol, UK), or GW1929 (PPARγ agonist; Sigma-Aldrich) at various concentrations or with 0.1% ethanol (vehicle control) at the beginning of day 1 of culture. The medium containing rmFlt3L and/or chemicals or 0.1% ethanol was refreshed on days 4 and 6. On day 8, the cells were stained with the following antibodies, for phenotype analysis using flow cytometry: PE-conjugated anti-CD86 (GL-1; eBioscience), PerCP-cy5.5-conjugated anti-CD24 (M1/69; BioLegend), BV421-conjugated anti-CD45RA (14.8; BD Biosciences), APC-conjugated anti-CD11c (N418), APCcy7-conjugated anti-CD11b (M1/70; BioLegend), Alexa Fluor™ 700-conjugated anti-MHC II (M5/114.15.2), and LIVE/DEAD™ Fixable Red. For functional analysis, day-8 FL-DCs were washed and then stimulated with CpG1826 (10 μg/ml, InvivoGen, Carlsbad, CA, USA) for 24 h. The supernatants of FL-DCs were assessed for levels of cytokines using ELISA (R&D Systems).