Phenol red free rpmi 1640

T47D-KBluc and MDA-MB-453 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The MCF7 and HeLa cell lines were a gift from Dr Sandra Dunn (Division of Hematology and Oncology, Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada). TamR cell lines TamR3 and TamR6 were kindly provided by Dr Euphemia Leung (University of Auckland, New Zealand) [45 (link)]. Cells were cultured at 37°C in a humidified incubator with 5% CO₂. The cell lines were maintained in the following culture media: for MCF7, phenol red–free RPMI 1640 (Gibco/Life Technologies, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS) (Gibco/Life Technologies); for T47D-KBluc, phenol red–free RPMI 1640 containing 4.5 g/L glucose (Sigma-Aldrich, St Louis, MO, USA), 10 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (Sigma-Aldrich), pH 7.5, 1 mM sodium pyruvate (Life Technologies), 0.2 U/ml insulin (Sigma-Aldrich) and 10% FBS; for MDA-MB-453 and HeLa, Dulbecco’s modified Eagle’s medium (HyClone Laboratories/GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% FBS (Gibco/Life Technologies); and for TamR3 and TamR6, phenol red–free RPMI 1640 containing 10% charcoal-stripped serum (CSS) (Gibco/Life Technologies) and 1 μM tamoxifen (Sigma-Aldrich).

The primary antibodies for NCAPD3 and PCLAF were obtained from Santa Cruz Biotechnology (CA, USA). Other primary antibodies for STAT3, AR, MYC, EZH2, AGO2, Ki67, β-actin and the secondary antibodies, including HRP Goat Anti-Mouse IgG and HRP Goat Anti-Rabbit IgG, were obtained from ABclonal Technology (Hubei, China). MALAT1-IN-1 and Stattic were purchased from MedChemExpress (NJ, USA). Mibolerone (Mib) was purchased from Sigma Aldrich (MO, USA). Charcoal-stripped serum (CSS) was purchased from Biological Industries (Beit Haemek, Israel). Phenol red-free RPMI 1640 was purchased from Gibco (New York, USA).

MCF-7 and T47D were obtained from Prof Yu-Mei Feng [99 (link),100 (link)]. The cells were grown in RPMI-1640 media (Biological Industries, Beit-Haemek, Israel) with 10% FBS (Biological Industries, Beit-Haemek, Israel) and 1% penicillin/streptomycin in a humidified atmosphere (95% air, 5% CO2) at 37 °C. To avoid background estrogenic stimulation, MCF-7 and T47D cells were cultured in hormone stripped medium (Phenol red-free RPMI1640 (#11835-030, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% charcoal-stripped fetal bovine serum (#04-201-1A, Biological Industries, Beit-Haemek, Israel) for 56 h, followed by treatment with 10⁻⁸ mol/L E2, P4 or MPA for 16 h.

RPMI-1640, fetal bovine serum, and phosphate buffered saline (PBS) were obtained from Lonza (Basel, Switzerland). Phenol red-free RPMI-1640, antibiotics, and Trypsin-EDTA were from Gibco BRL. The specific EGFR inhibitor BIBX1382 was from Calbiochem (La Jolla, CA). Goat antihuman p-PI3K antibody (p85a, Tyr508) was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antihuman EGFR antibody, mouse antihuman p-EGFR antibody (Tyr1068), ERK2 antibody, p-MAPK antibody (p44/p42, Thr202/Tyr204), rabbit antihuman PI3K antibody and horseradish peroxidase (HRP)-conjugated secondary anti-rabbit, anti-mouse, and anti-goat IgG antibodies were from Cell Signaling Technology (Danvers, MA). Rabbit antihuman GLUT1 antibody was from Dako. All other reagents were obtained from Sigma Chemicals (St. Louis, MO). The specific PI3K inhibitors wortmannin and LY294002, the specific MAPK inhibitor PD98059, the selective EGFR kinase inhibitor BIBX 1382 and gefitinib, and the protein synthesis inhibitor cycloheximide were prepared as stocks in dimethyl sulfoxide (DMSO). Doses used were 100 nM for cycloheximide, 5 μM for BIBX1382, 5 μM for gefitinib, 200 nM for wortmannin, 10 μM for LY294002, and 50 μM for PD98059. Ethanol and DMSO vehicles were used as controls as indicated.

RPMI 1640, fetal bovine serum (FBS), penicillin/streptomycin and trypsin were purchased from Gibco (Carlsbad, CA, USA). Recombinant visfatin was obtained from Peprotech (London, UK). Epidermal growth factor (EGF), 17-β estradiol (E2), FK866, LY294002, and U0126 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phenol red-free RPMI 1640, Opti-MEM I reduced serum medium and lipofectamine 2000 transfection reagent were purchased from Invitrogen Corporation (Carlsbad, CA, USA). Horseradish peroxidase-conjugated goat anti-rabbit IgG and polyclonal antibodies against human ERα, pERα (Ser118) and pERα (Ser167) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cignal ERE reporter assay kit and dual-luciferase reporter assay kit were purchased from Qiagen (Hilden, Germany) and Promega (Madison, WI, USA), respectively.