L glutamine

MCF-7 (breast cancer) and Ishikawa (endometrial cancer) ER-positive cancer cell lines were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). MCF-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Wako Pure Chemical Industries) supplemented with 10% fetal bovine serum (FBS; Nichirei Biosciences, Tokyo, Japan), 1% penicillin/streptomycin (Wako Pure Chemical Industries), and 2 mM L-glutamine (Life Technologies), and cultured at 37°C under a humidified 5% CO 2 atmosphere. Ishikawa cells were maintained in DMEM supplemented with 5% FBS, 1% penicillin/streptomycin, and 2 mM L-glutamine under the same atmospheric conditions as MCF-7 cells. Cells were plated onto a 24-well culture plate at 5 × 10 4 cells/well and allowed to attach overnight. The next day, the medium was replaced with phenol red-free DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% charcoal-stripped FBS (Equitech Bio, Kerrville, TX, USA), 1% penicillin/streptomycin, and 2 mM L-glutamine, and incubated for a day. Test chemicals were dissolved in DMSO and applied to cells at final DMSO concentrations of less than 0.1%. After 24 hr of chemical treatment, cells were collected and used immediately in downstream assays or stored at -80°C until use.

HepG2-AD13 cells, subclonal HepG2 cells stably transfected with an ARE-destabilized luciferase reporter vector (pGL4.24-2E-Neo), were previously established 15) . The vector pGL4.24-2E-Neo is a variant of the vector pGL4.24-2E and contains a neomycin-resistant gene (Neo), whereas the vector pGL4.24-2E contains 2 copies of ARE and a minimal TATA promoter (minP) immediately upstream of a synthetic firefly luciferase gene followed by a destabilization sequence 15) . HepG2-AD13 cells were incubated at 37°C under an atmosphere of 95% air and 5% CO 2 in Dulbecco's minimum essential medium (D-MEM) (Wako Pure Chemical Industries) containing 0.292 g/L L-glutamine, 10% heat-inactivated fetal bovine serum (Equitech-Bio, Kerrville, TX), and 500 µg/mL G418 (Wako Pure Chemical Industries). Confluent cultures (approximately 80%) were washed with phosphate-buffered saline without calcium and magnesium (PBS (-)), detached with 0.25% trypsin (Wako Pure Chemical Industries), and then sub-cultured.