Selenium its

The normal murine hepatocyte cell line, AML12 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). AML12 cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium (Cytiva/Hyclone, Marlborough, MA, USA), supplemented with 10% fetal bovine serum (FBS) (ThermoFisher, Waltham, MA, USA), 1% penicillin and streptomycin (ThermoFisher, Waltham, MA, USA), 0.5 mM sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA), 5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml selenium (ITS) (Sigma-Aldrich, St. Louis, MO, USA), 40 ng/ml dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), and 0.6 g of sodium bicarbonate (Daejung Co., Siheung, Korea). The cells were maintained at 37°C and in a 5% CO₂ humidified incubator.

DMCs were plated in 12-well plates (Corning, Tewksbury MA, USA) in a chondrogenic differentiation medium (DMEM-High Glucose, Thermo Scientific, Waltham, USA), as well as 1% penicillin and streptomycin (Gibco, Life Technologies, Carlsbad, CA, USA), 10^-7M dexamethasone (Sigma-Aldrich, MO, USA), 50μg/ml ascorbate- 2-phosphate (Sigma-Aldrich, MO, USA), 0.5mg/ml bovine serum albumin (Sigma-Aldrich, MO, USA), 5μg/ml linoleic acid (Sigma-Aldrich, MO, USA), 10mg/ml insulin, 5.5mg/l transferring, and 5μg/l selenium (ITS) (Sigma-Aldrich, MO, USA). In addition 10ng/ml transforming growth factor-β 3 (TGFβ3) (Sigma-Aldrich, MO, USA) was added. The samples were cultured for 14 days. The DMCs were then washed in PBS and fixed with 4% paraformaldehyde for 15 minutes. Cell staining was assessed by using 0.5% Toluidine Blue (Beyotime Institute of Biotechnology, ShangHai, China). The results were detected by microscopy.
Adipogenesis was induced in the presence of 2mg/ml insulin, 100mM isobutyl methyl xanthine (IBMX), 10mg/ml indomethacin, and 10⁻³M dexamethasone in the basal media of the DMCs. Adipogenic cells were stained after day 14 using Oil Red O (Millipore, Billerica, MA). The results were detected by microscopy.