Lm002 05

All cell culture reagents were purchased from Welgene (Gyungsan, Korea). Hep3B human hepatocyte cancer cell and Raw 264.7 mouse kupffer cell were maintained at 37 °C in a 5% CO2 atmosphere in DMEM (Welgene, LM001–05) supplemented with 5% (vol/vol) fetal bovine serum, penicillin (100 U/mol) and streptomycin (100 μg/ml). For experiments, cells were maintained in a steroid-free condition with phenol free DMEM (Welgene, LM002–05) media containing 2% charcoal dextran treated-FBS, penicillin (100 U/mol) and streptomycin (100 μg/ml). Genistein (LC laboratories, G-6055) and cobalt(II) chloride hexahydrate (Sigma, C8661) were added every 12 h with media change. Lipopolysaccharides (LPS, Sigma, L6511) was added after dissolved in distilled water (100 ng/ml). All cell experiments were repeated at least 3 times.

To validate in vivo experimental data, we performed in vitro assays using the H9c2 cardiomyocytes line. The cells were grown at 37 °C in a 5% CO₂ atmosphere and were cultured with Dulbecco’s modified Eagle’s medium (DMEM)-High glucose (LM 001-05, Welgene, Gyeongsan, Gyeongbuk, Korea) added to 5% fatal bovine serum (FBS) and 1% penicillin-streptomycin (P/S). Additionally, to remove endogenous steroid hormones, a starvation medium was used, composed of DMEM/F-12 (LM 002-05, Welgene) added to 2% charcoal dextran fetal bovine serum (CD-FBS) and 1% P/S.
To assess fluctuations in lipid metabolism, we supplied fatty acids to cells. Fatty acids (Palmitic acid 330 μM, Oleic acid 660 μM) dissolved in absolute ethanol (K46253383 506, EMD Millipore Corp.) were treated and incubated for 48 h. Letrozole, ethynylestradiol and Testosterone (Letrozole, L0248, Tokyo chemical industry CO. LTD., Tokyo, Japan) (Ethynylestradiol, E0037, Tokyo chemical industry CO. LTD) (Testosterone, T0027, Tokyo chemical industry CO. LTD) were dissolved in dimethyl sulfoxide (DMSO) (#MKCH9998, Sigma-Aldrich, St. Louis, MO, USA). The three reagents were added to 100 nM/L respectively and incubated for 24 h.