Fetal bovine serum (fbs)

The human oral squamous cell carcinoma cell line HSC-2 (RCB1945; RIKEN BRC, Ibaraki, Tsukuba, Japan) was cultured in EMEM cell culture medium (FUJIFILM Wako) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin G and 100 U/mL streptomycin; FUJIFILM Wako) under cell culture conditions (37 °C, 5% CO₂ atmosphere).

PDL cells of LepR-cre; R26-tdTomato mice were cultured at 37 °C and 5% CO₂ in 96-well plates (1.5 × 10⁵ cells/well) (Corning) up to subconfluent density with 200 μl DMEM (10567-014) containing 1% Antibiotic–Antimycotic and 10% mesenchymal stem cell-qualified FBS (12662-011) (all from Thermo Fisher Scientific); in case of osteoblastic differentiation, the 96-well plats were coated with 10% Cellmatrix (Type I-A) (Nitta Gelatin Inc., Osaka, Japan). For osteoblastic differentiation, cells were cultured with αMEM (M4526) containing 5 mM β-glycerophosphate, 100 μg/mL ascorbic acid (all from Sigma-Aldrich), 10% FBS (Fujifilm Wako Pure Chemical), 1% Antibiotic–Antimycotic, 2 mM L-Glutamine (both from Thermo Fisher Scientific), and 200 ng/mL BMP2 (R&D SYSTEMS) for 1 week. Medium was changed every 2–3 days. Adipocytic differentiation was induced using AdipoInducer Reagent (for animal cell) (Takara) kit, according to the manufacturer’s instructions. Briefly, cells were cultured with RPMI 1640 medium containing 10% FBS (both from Fujifilm Wako Pure Chemical), 1% Antibiotic–Antimycotic (Thermo Fisher Scientific), insulin (10 μg/mL) and dexamethasone (2.5 μM) at 37 °C and 5% CO₂ for 48 h; the medium was replaced with RPMI 1640 medium containing 10% FBS, 1% Antibiotic–Antimycotic and insulin (10 μg/ml) and cultured at 37 °C and 5% CO₂ for 2–3 weeks.

Cells were cultured with each medium or condition: Dulbecco’s modified Eagle medium (DMEM) with high glucose 4500 mg/L and 16 amino acids (WAKO, Osaka, Japan) and 10% Fetal bovine serum (FBS; SIGMA, St. Louis, MO) as control medium, DMEM with low glucose 1000 mg/L and 16 amino acids and 10% FBS as low-glucose medium, DMEM with high glucose 4500 mg/L and no amino acids (WAKO) and 10% FBS as amino-acid-free medium, DMEM with high glucose 4500 mg/L and 16 amino acids, without FBS as FBS-free medium. These media were also contained penicillin and streptomycin, and 0.5 mM sodium pyruvate, and were incubated at 37 °C under 21% oxygen condition. Another condition was hypoxia: the control medium under 1%O₂. This study was approved by the Osaka City University ethics committee (approval number 924).