Phtpp

Human GBM-derived cell lines U87, U251, T98, and LN229 (American Type Culture Collection, ATCC, Manassas, VA, USA) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, L0107-500) high glucose supplemented with 10% fetal bovine serum (FBS; S1650), 1.0 mM pyruvate (L0642-100), 1.0 mM antibiotic (streptomycin 10 g/L; penicillin G 6.028 g/L; and amphotericin B 0.025 g/L, L0010), and 0.1 mM non-essential amino acids (X0557-100, Biowest, Nuaillé, PDL, France). Cell cultures were maintained at 37 °C in a humidified atmosphere with 5% CO₂. At 60% confluence (24 h before treatments), cells were culture in DMEM no phenol red (ME-019 Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% charcoal/dextran-treated FBS (SH30068.03, Thermo Fisher Scientific), 1.0 mM pyruvate, 1.0 mM antibiotics, and 0.1 mM non-essential amino acids. When indicated, cells were treated with E2 (10 nM, E4389, Sigma-Aldrich, St. Louis, MO, USA), ER-α-selective agonist PPT (10 nM, 1426, Tocris, Bristol, UK, England), ER-β-selective agonist diarylpropionitrile (DNP, 10 nM, 1494, Tocris), ER-α-selective antagonist MPP (1 μM, 1991, Tocris), and ER-β-selective antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazole[1,5-a]pyrimidin-3-yl]phenol (PHTPP, 1 μM, 2662, Tocris). In combined treatments, antagonists MPP and PHTPP were added 2 h before the addition of agonist.

PNT1a cells were obtained from M. Littmann (Baylor College of Medicine, Houston). The human prostate cancer cell line, LNCaP was obtained from American Type Culture Collection (ATCC). PC3-M cells were obtained from R. C. Bergan (Northwestern University, Chicago). 3β-androstane-diol (3β-Adiol) and PHTPP experiments were performed by incubating cells with 3β-Adiol (5 μM; Sigma) or PHTPP (10 μM; Tocris) for 2–3 days. The generation of ERβ-ablated PNT1a cells, PHD2-ablated PNT1a cells and HIF-1α ablated cells has been described previously [9 (link)]. IKKβ ablated PC3-M cells were generated using shRNAs (Open Biosystems; TRCN0000018918 and TRCN0000018919). Stable cell transfectants were generated by puromycin or hygromycin selection (0.5 μg/mL for PNT1a and 2 μg/mL for PC3-M cells). The resultant ERβ, HIF-1α-ablated cells were used for subsequent experiments. For experiments involving hypoxia, cells were incubated with 100 μM cobalt chloride for 22–24 hours.

The MDA-MB-231, HCC1806, and BT549 cell lines were obtained from the American Type Culture Collection. The SUM159 cell line was obtained from Thomas C. MDA-MB-231, HCC1806, and BT549 cells were maintained in RPMI-1640 (Invitrogen Inc., Carlsbad, CA, USA) medium supplemented with 10% fetal bovine serum (FBS) (PEAK, Wellington, CO, USA), and antibiotic-antimycotic (Invitrogen Inc., Carlsbad, CA, USA). The SUM159 cell line was maintained in DMEM (Invitrogen Inc., Carlsbad, CA, USA) medium supplemented with 10% fetal bovine serum (FBS) (PEAK, Wellington, CO, USA), and anti-anti (Invitrogen Inc., Carlsbad, CA, USA). All experiments used cells below passage 30. ERβ antagonist PHTPP (Tocris, Minneapolis, MN, USA). Paclitaxel was purchased from Millipore (St. Louis, MO, USA). MTS reagent was from Biovision (Milpitas, CA, USA).

Bisphenol-A was obtained from MP Biomedicals (Cat No 155118; Santa Ana, CA, USA). 17β-oestradiol (Cat No E8875), diazoxide (Cat No D9530), and nifedipine (Cat No D7634) were obtained from Sigma-Aldrich (Saint Louis, MO, USA). SNX-482 (Cat No 4363) was obtained from PeptaNova (Sandhausen, Germany). DPN (Cat No 1494), PPT (Cat No 1426), PHTPP (Cat No 2662), MPP dihydrochloride (Cat No 1991), PD98059 (Cat No 1213), and Wortmannin (Cat No 1232) were obtained from Tocris Cookson (Bristol, UK).

E15 mouse embryos were classified according to the sex or/and genotype. Cultured amygdala neurons were prepared and maintained as described^{7 (link)}. The medium was phenol red-free Neurobasal supplemented with B-27, N-2 and GlutaMAX I (Invitrogen). After 3 DIV the culture medium was replaced for 2 h by fresh medium devoid of supplements for Western blotting and quantitative real-time polymerase chain reaction analysis. Neuronal cultures were treated for 2 h with the following test compounds alone or in combination: E2 (10⁻¹⁰ M; Sigma-Aldrich), DHT (10⁻¹⁰ M; Steraloids), 3β-diol (10⁻¹⁰ M; Sigma-Aldrich), the selective ERα agonist propylpyrazoletriol (PPT, 10⁻⁷ M; Tocris Bioscience), the selective ERβ agonist dyarilpropionitrile (DPN, 10⁻⁹ M; Tocris), the GPER agonist G1 (10⁻⁷ M, Calbiochem), the selective ERβ antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP, 10⁻⁹ M; Tocris) and the selective AR antagonist flutamide (10⁻⁷ M; Tocris).