Trout serum

ZFL cells were cultured as previously reported [21 (link)]. Briefly, cells were cultured in a mixed medium containing 50% L-15, 35% DMEM HG, and 15% Ham’s F12 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), which was supplemented with 0.15 g/L sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), 15 mM HEPES (Sigma-Aldrich), 0.01 mg/mL bovine insulin (Sigma-Aldrich), 50ng/mL epidermal growth factor (Sigma-Aldrich), 5% fetal bovine serum (Gibco), and 0.5% trout serum (Caisson Labs, Smithfield, UT, USA). The cells were cultured in a cell incubator set at 28 °C.
For the LD formation experiment, the cells were incubated either with NM or with high-fat medium (HFM) containing 300 μM bovine serum albumin (BSA)-coated oleic acid (Sigma-Aldrich) for 6 h. The BSA-coated oleic acid is obtained by adding the oleic acid dissolved in ethanol dropwise to the BSA solution to obtain a homogenous mixture. For the retinyl acetate treatment experiment, 10 μM retinyl acetate (MedChemExpress, Monmouth Junction, NJ, USA) was added to cells cultured in NM or HFM, followed by incubation for 6 h.

The zebrafish liver (ZFL) cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in a mixed medium (50% L-15, 35% DMEM HG, and 15% Ham’s F12; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 0.15 g/L sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), 15 mM HEPES (Sigma-Aldrich), 0.01 mg/mL bovine insulin (Sigma-Aldrich), 50 ng/mL epidermal growth factor (Sigma-Aldrich), 5% fetal bovine serum (Gibco), and 0.5% trout serum (Caisson Labs, Smithfield, UT, USA). The cells were incubated at 28 °C in a 100% air atmosphere. The medium was replaced every 2 days.
LD-containing cells were prepared by adding 400 μM lipid mixture (200 μM oleic acid, 100 μM linoleic acid, and 100 μM linolenic acid; coated with bovine serum albumin; Sigma-Aldrich) for 24 h. LD-containing cells were directly incubated with PGF2α and PGE2 (MedChemExpress, Monmouth Junction, NJ, USA; dissolved in dimethyl sulfoxide). Before treatment, cytotoxicity of the molecules was assessed using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay.

Fetal bovine serum (FBS), Leibowitz’s L-15 medium, the Roswell Park Memorial Institute (RPMI) 1640 medium, Ham’s F12 medium, Eagle’s minimal essential medium (EMEM), Dulbecco’s modified Eagle’s medium (DMEM), the Opti-MEM medium, a penicillin/streptomycin solution and trypsin were obtained from GIBCO (Grand Island, NY, USA). Trout serum was purchased from Caisson Laboratories (Smithfield, VA, USA). Mouse epidermal growth factor (EGF) and HEPES were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Sodium bicarbonate, bovine insulin, DHT, DHT-D3 solution, methyl-tertiary-butyl ether (MTBE), trimethylamine (TEA), tetrahydrofuran (THF), 2- PA, 4-(dimethylamino) pyridine (DMAP), 2-methyl-6-nitrobenzoic anhydride (MNBA), and acetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA), and HPLC-grade formic acid was purchased from Fisher Scientific (Pittsburgh, PA, USA). MS-grade methanol and water were obtained from VWR (Westchester, NY, USA). The stock solution and internal standard were prepared in methanol. The derivatization reagent was prepared by dissolving 25.0 mg of PA, 10.0 mg of DMAP, and 20.0 mg of MNBA in 1 mL of THF (Yamashita et al., 2009) and vortexing. Then, the mixture was left at room temperature for at least 5 min before the sample pretreatment.