4 androstenedione

Reagents were obtained from the following sources: 17-β-estradiol (E2), 4-hydroxytamoxifen and 4-androstenedione (Sigma-Aldrich); Fulvestrant (Tocris); Linsitinib (MedChemExpress) everolimus, pictilisib, palbociclib and abemaciclib (SelleckChem). Elacestrant was supplied by Radius Health Inc.

All hormones, selective estrogen-receptor modulators (SERMS) were obtained from Sigma-Aldrich: 4-andro-stenedione (A9630), cholesterol (C8667), corticosterone (C2505), estradiol (E1024), estriol (E1253), estrone (E1274), hydrocortisone (H3160), 5-Pregnen-3β-ol-20-one (P9129), progesterone (P3972), Raloxifene HCl (R1402), Tamoxifen citrate salt (T9262), and Genistein (92136). We did not test testosterone since our department lacks the approval to obtain this controlled substance.

In order to study their steroidogenic function, COV434 and hGC were cultivated in 24-well plates (Sigma-Aldrich, St. Louis, MO, EUA. SIAL0526) at a density of 30 × 10⁴ cell/well and 70 × 10⁴ cell/well, respectively. On the next day, RES was added at a final concentration of 0.01 and 5 μM. In addition, 1 unit of follicle-stimulating hormone (FSH) and 4-androstenedione (Sigma-Aldrich, USA), dissolved in DMSO, were added. After 72 h of incubation, cell culture media were collected, centrifuged, and stored at –80 °C. VIDAS^® Estradiol II kits (bioMérieux SA, Marcy l’Etoile, France) were used to study the estradiol secretion by resorting to the enzyme-linked fluorescent assay (ELFA). DNA isolation was performed using TripleXtractor reagent, (GRiSP Research Solutions, Porto, Portugal), and quantified in the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies,Inc., Wilmington, DE, USA). Hormone levels were normalized to cell DNA.