Sb204990

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.