Bicalutamide bic

The mouse Sertoli cell lines TM4 (Cat no. CRL-1715) and 15P-1 (Cat no. CRL-2618) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained under standard conditions. Properties of both cell lines were evaluated according to cell line authentication recommendations of the Global Bioresource Center (ATCC). Microscopic observation, analysis of proliferation and viability and detection cell-specific markers expression (androgen binding protein (Abp), Desert Hedgehog (Dhh), SRY-box 9 (Sox9), sulphated glycoprotein-2 (Sgp-2), GATA binding protein 4 (Gata4); Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland; Table S1) were performed. Cells were seeded directly into cell culture plates or on coverslips. Before each experiment, cells were serum starved for 24 h. The same cell concentration (0.5 × 10⁶/cm²) was used in all experiments, so the ratio of T concentration and initial cell number was constant between experiments.
Cells were treated with 10⁻⁸ M to 10⁻⁶ M testosterone (T, Cat no. 86500; Sigma-Aldrich, St. Louis, MO, USA) or anti-androgens 10⁻⁴ M hydroxyflutamide (HF; Cat no. H4166; Sigma-Aldrich) or 10⁻⁶ M bicalutamide (Bic) (Cat no. B9061; Sigma-Aldrich) alone or with the addition of 10⁻⁸ M T for 24 h. Control cells were incubated in the presence of the vehicle (0.01% dimethyl sulfoxide, DMSO).

The androgen-dependent (AD) PCa cell lines 22RV1 and Vcap were purchased from the American type culture collection (Manassas, VA, USA), and the androgen-independent (AI) PCa cell lines, PC3 and DU145, were obtained from the cell bank of the Chinese academy of sciences (Shanghai, China). The cell lines were cultured in RPMI-1640 media (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS, Invitrogen) at 37 °C in a humidified atmosphere with 5% CO₂. 5α-dihydrotestosterone (DHT) and bicalutamide (BIC) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

LNCaP cells were cultured in 6‐well plates at a density of 4 × 10⁵ cells/well in hormone‐depleted medium, comprising phenol red‐free RPMI (Sigma) supplemented as above but with 5% charcoal‐stripped fetal bovine serum (Labtech International), for 72 h prior to treatment with 10 nM mibolerone (Mib) (PerkinElmer Life Sciences, Wellesley, MA), 1 μM bicalutamide (Bic) (Sigma, Chemical Co., St. Louis, MO) or 10 nM 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG) (Sigma). Cells were harvested for protein extraction and protein expression assessed by Western blotting using 10 μg of total lysate.