Fulvestrant

A549 and H1793 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and grown for 2 weeks (five passages). A549 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO₂. H1793 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium supplemented with 5% FBS, 0.005 mg/ml insulin, 0.01 mg/ml transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, and extra 2 mM l-glutamine at 37°C with 5% CO₂. For experiments, cells were plated in phenol red-free medium that was supplemented with either charcoal-stripped serum or no serum for at least 24 h prior to adding the ligand, and 48 h later they were treated with the following agents: E2 (Sigma-Aldrich, St. Louis, MO, USA), (1-(4-(6-bromobenzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)-ethanone, 1-[(3aS,4R,9bR-rel)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone) (G1, a GPER agonist; Cayman Chemical, Ann Arbor, MI, USA), fulvestrant (an ER antagonist and also a GPER agonist; Cayman Chemical), and (4-(6-bromo-benzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quinoline) (G15, a GPER antagonist; Cayman Chemical) at the indicated concentrations. For cell culture experiments, all reagents used were formulated in 100% dimethyl sulfoxide.