Foetal bovine serum (fbs)

The human cervical cancer cell line HeLa (RIKEN Cell Bank) and human prostate cancer cell line LNCaP (RIKEN Cell Bank) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Nacalai Tesque) or RPMI-1640 (Nacalai Tesque) with 10% (v/v) foetal bovine serum (Nichirei Biosciences) and 1% (v/v) penicillin–streptomycin (Life Technologies) and incubated at 37 °C and 5% CO₂ in static culture.
The insect cell line sf9 (Thermo Fisher Scientific) was maintained in Grace’s Insect Medium (Thermo Fisher Scientific) with 10% (v/v) foetal bovine serum and 1% (v/v) penicillin–streptomycin (Life Technologies) and incubated at 27 °C.

All experimental protocols involving the use of dogs were approved by the Bioethics Committee at Nippon Veterinary and Life Science University. Six healthy beagles (three males and three females; mean age 1.5 years; mean body weight 9.2 kg) (ORIENTAL YEAST, Tokyo, Japan) were included in this study.
Adipose tissue was aseptically collected from the falciform ligament fat of the six anaesthetised dogs. The tissue was washed extensively with PBS, minced, and digested with collagenase type I (Sigma-Aldrich, St. Louis, MO) at 37°C for 45 min with intermittent shaking. After washing with PBS and centrifuging, the pellets, containing the stromal vascular fraction, were resuspended, filtered through 100-μm nylon mesh and incubated overnight in high glucose Dulbecco’s Modified Eagle’s medium (H-DMEM) supplemented with 10% foetal bovine serum (FBS; Nichirei Bioscience, Tokyo, Japan) and 1% antibiotic–antimycotic (Thermo Fisher Scientific, Waltham, MA) in a humidified atmosphere of 5% CO₂ at 37°C. Unattached cells were removed by changing the medium, and the attached cells were washed twice with PBS. Thereafter, the medium was replaced every 3–4 days. When the cells reached 80%–90% confluence, they were detached with trypsin-EDTA solution (Sigma-Aldrich, St. Louis, MO) and passaged.

L‐Ornithine, sodium benzoate, 5‐azacytidine, NaPB, resveratrol, Vitamin K2 and ammonium chloride were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Other collections of small compounds acting as dopamine D3 receptor inhibitor (43 compounds), targeting mammalian targets of rapamycin (mTOR) pathway (58 compounds), or tumour necrosis factor (TNF) pathway (76 compounds) were synthesized and provided by Dr. Wu Zhong's laboratory from the Beijing Institute of Pharmacology & Toxicology. Dulbecco's modified Eagle's medium (DMEM, with or without Phenol Red) was purchased from Gibco (Grand Island, NY, USA). Foetal bovine serum (FBS) was purchased from Nichirei Biosciences (Tokyo, Japan). Hochest 33342, MitoTracker^® Green FM^® and lipofectamine^® 2000 were purchased from Invitrogen (Carlsbad, CA, USA).

The affinity of the cultured hepatic cells for saturated and unsaturated fatty acids was evaluated by the methods previously described by Gómez-Lechón et al.28 (link) and Hozumi et al.29 (link) with some modifications. First, 1 mM of each fatty acid was incubated for 24 h with the cultured hepatic cells (HepG2 cells derived from human hepatic cancer cells; DS Pharma Biomedical Co., Ltd., Osaka, Japan) in Dulbecco’s modified Eagle’s Medium (DMEM) (Sigma-Aldrich, Tokyo, Japan) containing foetal bovine serum (FBS) (Nichirei Biosciences Inc., Tokyo, Japan), which is lipid- and fatty acid-free, for 24 h. The mixtures were then washed twice with PBS, fixed with 10% formalin for 10 min at the room temperature, and then washed twice more. Next, 1 ml of 60% isopropanol was added and incubated on the samples for 1 min before being discarded, after which 1 ml of Oil Red O staining solution was added and incubated with the samples for 20 min at room temperature. Final washes were conducted, using 60% isopropanol once, followed by PBS twice. Finally, the culture dishes were examined by light microscopy (Inverted Microscope Olympus CKX41, Olympus, Osaka, Japan).