Bt 474

The breast cancer cell lines MCF-7, T47D, ZR-75-1, BT-474, SKBR3, and MDA-MB-231 were obtained from RIKEN Bioresource Research Center (Tsukuba, Japan). The cells were maintained in phenol red-free Dulbecco’s modified Eagle’s medium (DMEM) (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) containing 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Fujifilm Wako Pure Chemical Corporation) at 37 °C with 5% CO₂. For time-series metabolomics, 3.5 × 10⁵ cells were seeded in 3.5 mL of medium in 60-mm-diameter dishes. After a 15-h incubation, following rapid washing twice with FBS-free media, cells were cultured in 3.5 mL DMEM lacking glucose, l-glutamine, phenol red, sodium pyruvate, and sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 20 mM glucose, 2.0 mM glutamine, 3.7 g/L sodium bicarbonate, and 10% dialyzed FBS (Life Technologies, Carlsbad, CA, USA). [U-¹³C₅] Glutamine was used for isotope tracing experiments. The cells were treated with (Z)-4-hydroxytamoxifen (Sigma-Aldrich, St. Louis, MO, USA) in ethanol and SB204990 (MedChemExpress, Monmouth Junction, NJ, USA) or A922599 (MedChemExpress) in dimethyl sulfoxide (DMSO). In all experiments, control cell cultures were treated with an equivalent volume of the corresponding solvent.

Human breast cancer cell lines Hs578T, MDA-MB-231, MCF-7, and BT-474 and the human mammary epithelial cell line H184B5F5/M10 were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). These cell lines were cultured in Dulbecco’s Modified Eagle Medium (Sigma, St. Louis, MO, USA) with 0.1 mM sodium pyruvate, 10% fetal bovine serum (Gibco, BRL, Grand Island, NY, USA), 2 mM l-glutamine, 100 U mL⁻¹ penicillin, and 100 µg mL⁻¹ streptomycin (Sigma-Aldrich, Saint Louis, MO, USA). The cells were maintained at 37 °C in a humidified incubator with 5% CO₂.

MCF-7 (luminal A), MDA-MB-231 (triple-negative), and BT-474 (luminal B) human breast carcinoma cells and HS68 human foreskin fibroblast cells were obtained from the Bioresource Collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsinchu, Taiwan). MCF-7, MDA-MB-231, and HS68 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen Life Technologies, CA, USA) and BT-474 cells were cultured in RPMI (Invitrogen). All media used contained 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen). The cells were incubated at 37°C in an atmosphere containing 5% CO₂.