Antibiotic antimycotic

CHO-S cells were cultured in Power-CHO medium (Lonza), containing 8 mM Ultraglutamine (Lonza), 4 mM HT-supplement and 1× antibiotic-antimycotic (Gibco) at 37°C. The cells were maintained at densities between 0.5 and 8.0 × 10⁶ cells/mL in a shaking incubator (150 rpm).
In general, the remaining cancer cells were cultured under a 5% CO₂ atmosphere at 37°C. CTLL-2 and transduced reporter CTLL-2 cells [45 (link)] were cultured in RPMI (Gibco) containing 10% FBS (Gibco), 1× antibiotic-antimycotic (Gibco), 2 mM Ultraglutamine (Lonza), 25 mM HEPES (Gibco), 50 μM β-mercaptoethanol (Sigma) and 60 U/mL IL-2 (Proleukin, Roche Diagnostics). F9 teratocarcinoma cells were cultured in flasks coated with 0.1% gelatin (Type B from Bovine Skin, Sigma), and they were cultured in DMEM (Gibco) containing 10% FBS (Gibco) and 1× antibiotic-antimycotic (Gibco). WEHI-164 fibrosarcoma cells were cultured in RPMI (Gibco) containing 10% FBS (Gibco) and 1× antibiotic-antimycotic (Gibco).

SUM159 and SUM149 BC cells were cultured in Ham’s F-12 (ThermoFisher Scientific) supplemented with 5% FBS (ThermoFisher Scientific), 5 µg/mL insulin (Sigma-Aldrich, St. Louis, MO), 1 µg/mL hydrocortisone (Sigma-Aldrich), and 1x antibiotic-antimycotic (ThermoFisher Scientific, 100x). MCF7 cells were grown in EMEM medium (ATCC) supplemented with 10% FBS, 1x antibiotic-antimycotic, and 10 µg/mL insulin (Sigma-Aldrich). BT20, MDA-MB-231, MDA-MB-468, MDA-MB-157 and SKBR3 were cultured in DMEM-high glucose (Gibco) supplemented with 10% FBS and 1x antibiotic-antimycotic. Vari068, HCC38, HCC70, HCC1937, HCC1954, T47D, ZR-75-1 and BT474 were maintained in RPMI1640 medium (ThermoFisher Scientific) supplemented with 10% FBS and 1x antibiotic-antimycotic. MCF10A is cultured in DMEM/F12 media (50:50, ThermoFisher Scientific) supplemented with 5% horse serum, 1x HEPES, 20 ng/ml EGF, 0.5 mg/ml hydrocortisone, 100 ng/ml cholera toxin, 10 µg/ml insulin and 1x antibiotic-antimycotic. All the cell lines are cultured at 37 °C under 5% CO₂ in a humidified chamber and are mycoplasma-free.

Mouse hepatoma (Hepa-1c1c7) and mouse normal liver (NCTC clone 1469) cell lines were obtained from KCLB (Korean Cell Line Bank, Seoul, Korea). Human hepatoblastoma cell line (HepG2) and human colon carcinoma cell line (HCT116) were obtained from the American Type Culture Collection (ATCC, VA, USA). Hepa-1c1c7 cells were maintained in minimum essential medium (MEM) (WelGENE Inc., Daegu, Korea) supplemented with 10% fetal bovine serum (FBS) (HyClone Inc., Logan, UT) and 1% antibiotic-antimycotic (Gibco, Grand Island, NY, USA). HepG2 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) (WelGENE Inc.) containing 10% FBS (HyClone Inc.) and 1% antibiotic-antimycotic (Gibco). HCT116 cells were maintained in McCoy’s 5A medium (WelGENE Inc.) containing 10% FBS (HyClone Inc.) and 1% antibiotic-antimycotic (Gibco). NCTC clone 1469 cells were maintained in DMEM (WelGENE Inc.) containing 10% horse serum (HyClone Inc.) and 1% antibiotic-antimycotic (Gibco). Cells were maintained at 37 °C in a humidified atmosphere containing 5% of CO₂.

Differentiation of 3T3-L1 preadipocytes into mature adipocytes was performed as previously described^{56 (link)}. In brief, 2-day postconfluent cells (designated day 0) were treated with DMEM, 10% FCS, 1% Antibiotic-Antimycotic (Gibco®), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.25 µm Dexamethasone, 1 µg/ml insulin and 2 µM Rosiglitazone (all Sigma-Aldrich) to induce differentiation. After 3 days, medium was replaced by DMEM, 10% FCS, 1% Antibiotic-Antimycotic (Gibco®) and 1 µg/ml insulin. On day 6, medium was replaced with regular culture medium (DMEM, 10% FCS, 1% Antibiotic-Antimycotic), and further exchanged every 2-3 days. 3T3-L1 adipocytes were used for co-cultures with human breast cancer cells from day 10 to 18 after starting differentiation. 3T3-L1 preadipocyte control cells (undifferentiated control) were maintained in DMEM supplemented with 10% FCS and 1% Antibiotic-Antimycotic (Gibco®).
To generate adipocyte-conditioned medium (ACM), 3T3-L1 adipocytes (between day 10 and 18 after starting differentiation) were cultured in DMEM, 10% FCS, 1% Antibiotic-Antimycotic (Gibco®) for 48 h. ACM was then collected and filtered using a 0.8 µm syringe filter to remove any cellular debris.