Calf bovine serum

Mouse 3T3‐L1 preadipocytes were purchased from DS Pharma Biomedical. Preadipocytes were cultured in Dulbecco's modified Eagle's medium–high glucose medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% calf bovine serum (Gibco) and penicillin–streptomycin (Nacalai Tesque) at 37°C in a humidified 5% CO₂ atmosphere until confluence was reached in a 96‐well plate format. Two days after confluence (day 0), cells were stimulated to differentiate by culturing in adipocyte differentiation medium (ADM; DS Pharma Biomedical) for 3 days. Cells were then maintained in adipocyte maintenance medium (DS Pharma Biomedical) for an additional 4 days. Extracts or fractions (10 μg/ml) were administered from day 0 of adipocyte differentiation. On day 7, intracellular lipid droplets were stained using AdipoRed Assay Reagent (Lonza) according to the manufacturer's instructions. After obtaining images using a BZ‐X710 fluorescence microscope (Keyence), intracellular lipid accumulation was quantified by measuring fluorescence (Ex 485 nm/Em 590 nm) using a Victor2 multilabel plate reader (PerkinElmer).

3T3-L1 murine white preadipocytes (ATCC, Manassas, VA, USA) were grown up to the contact inhibition stage and remained in the post-confluent stage for 2 days in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% calf bovine serum (Gibco, Gaithersburg, MD, USA) and penicillin-streptomycin (Welgene, Daegu, Korea). Differentiation was then induced by changing the medium to DMEM supplemented with 10% FBS (Welgene) plus a cocktail of hormones (MDI) that include 0.5 mM IBMX (M) (Sigma, St. Louis, MO, USA), 0.5 μM dexamethasone (D) (Sigma) and 5 μg/mL insulin (I) (Sigma) in the presence or absence of tanshinone IIA at the indicated concentrations. After 48 h MDI-induction, the differentiation medium was replaced with DMEM supplemented with 10% FBS and 5 μg/mL insulin in the presence or absence of tanshinone IIA at the indicated concentrations. The cells were then fed every other day with DMEM containing 10% FBS in the presence or absence of tanshinone IIA at the indicated concentrations until day eight. On day eight, the preadipocytes became mature adipocytes that rounded-up and filled with many oil droplets.

H1299, 293FT, and MEF cells were maintained in DMEM containing 10% FBS (Corning Cellgro) and 1% penicillin/streptomycin (Gibco, NY, USA). PC-3 cells were cultured in RPMI (Hyclone) supplemented with 10% FBS (Corning Cellgro). NIH 3T3-L1 cells were maintained in DMEM (Gibco) with 10% calf bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). We purchased the 293FT cell line from Invitrogen, and the 3T3-L1 and H1299 cell lines were purchased from ATCC (American Type Culture Collection). PC-3 cells were provided by Kyung-sup Kim (Yonsei University College of Medicine, Seoul).

Briefly, 3T3-L1 pre-adipocytes cell line (60159, Bioresource Collection and Research Center, Taiwan) was seeded to a 12-well culture plate with a cell density of 1 × 10⁴ per well and cultured at 37°C under 5% CO₂ in Dulbecco Modified Eagle Medium (DMEM, high glucose, 12800-017, Gibco, United States) supplemented with 10% calf bovine serum (16170-078, Gibco, United States) and 1% of 100X Anti-anti. After confluence, it were further cultured in starvation condition for 2 days to keep cells in the status of G₀/G₁ phase at least 85% in all population (Cao et al., 2012 (link)). The confluent 3T3-L1 cells were cultured in an adipo-differentiated medium to convert cells into adipocytes; where the adipo-differentiated medium was DMEM supplemented with 10% FBS, 1% of 100X Anti-anti, 1 mM dexamethasone (D4902, Sigma, United States), 0.2 M indomethacin (I7378, Sigma, United States), 0.1% insulin and 0.25 M 3-Isobutyl-1-methylxanthine (IBMX, I5879, Sigma, United States). The adipocytes were cultured in DMEM supplemented 10% FBS and 1% of 100X Anti-anti; and medium was refreshed every 3 days, until the oil droplets were observed by a fluorescence microscope (TS-100, Nikon, Japan) stained with Nile red (N1142, Invitrogen, United States) (Park et al., 2017 (link)).

3T3-L1 preadipocytes (ATCC, Manassas, VA) were grown up to the contact inhibition stage and remained in the post-confluent stage for 2 days in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf bovine serum (Gibco, Grand Island, NY) and penicillin–streptomycin (Welgene, Daegu). Differentiation was then induced by changing the medium to DMEM supplemented with 10% fetal bovine serum (FBS) (Welgene, Daegu) plus a cocktail of hormones (MDI) that include 0.5 mM 3-isobutyl-1-methylaxanthine (M) (Sigma), 0.5 µM dexamethasone (D) (Sigma), and 5 µg/ml insulin (I) (Sigma) in the presence or absence of KMU-3 at the indicated concentrations. After 48-h (day 2) MDI-induction, the differentiation medium was replaced with DMEM supplemented with 10% FBS and 5 µg/ml insulin in the presence or absence of KMU-3 at the indicated concentrations. The cells were then fed every other day with DMEM containing 10% FBS in the presence or absence of KMU-3 at the indicated concentrations until day 8. On day 8, the preadipocytes became mature adipocytes that rounded-up and filled with many oil droplets.