# Molecular Targets of Minor Cannabinoids in Breast Cancer: In Silico and In Vitro Studies
## 4 Materials and Methods
### ER and AR Transactivation Assays  
Considering the molecular docking results, the activity of the nine selected cannabinoids towards human ER and AR was assessed as previously described [44; 100], and according to the guidelines from The Organization for Economic Cooperation and Development (OECD) for Testing of Chemicals, Tests No. 455 and 458, respectively. These bioassays are based on stably transfected mammalian cell lines and validated for a reliable detection of human ER and AR agonists and antagonists. Regarding the ER assay, we used the VM7 Luc4E2 cells, which were kindly provided by [PERSON] (University of California, USA). These cells express both human ER\(\alpha\) and ER\(\delta\) isoforms and were cultured in DMEM without phenol red, supplemented with 4.5% CFBS, 1% penicillin/streptomycin, 2% L-glutamine, and 110 mg/mL sodium pyruvate for 3 days before the beginning of the experiments, and then plated at a 4 \(\times\) 10\({}^{5}\) cells/mL cell density in white 96-well plates. After adhesion, cells were treated with the cannabinoids (1-10 \(\upmu\)M) for 24 h, in the absence (ER agonism) or presence of 91.8 pM 17\(\beta\)-estradiol (E\({}_{2}\); ER antagonism) or 1 nM testosterone (T; ER antagonism). The agonistic and antagonistic activities were measured using the Steady-Glo\({}^{\circledR}\) Luciferase Assay System (Promega Corporation, Madison, WI, USA) in a multimode plate reader (EnSpire\({}^{\circledR}\), Perkin Elmer, Inc., Waltham, MA, USA). Cell viability was assessed with the CellTiter-Glo\({}^{\circledR}\) Luminescent Cell Viability Assay (Promega Corporation, Madison, WI, USA).  
For AR activity assessment, the AR-EcoScreen\({}^{\text{TM}}\) cell line (#)CRB1328, from Japanese Collection of Research Bioresources Cell Bank, Tokyo, Japan) applied was based on Chinese hamster ovary (CHO-K1) cells expressing the human AR with a firefly luciferase reporter construct inserted and a renilla luciferase gene for the simultaneous evaluation of viability. Cells were seeded at a density of 9 \(\times\) 10\({}^{4}\) cells/mL in a DMEM/F12 medium without phenol red, containing 5% CFBS and 1% penicillin/streptomycin, in white 96-well plates. After adhesion, cells were exposed to the cannabinoids (1-10 \(\upmu\)M) for 24 h, in the absence (AR agonism) or presence of 0.1 nM methyltrienolone (R1881; AbMole BioScience, Houston, TX, USA) for investigating AR antagonism. Both AR activity and cell viability were assessed using the Dual-Glo\({}^{\circledR}\) Luciferase Assay System (Promega Corporation, Madison, WI, USA) in a multimode plate reader (EnSpire\({}^{\circledR}\), Perkin Elmer, Inc., Waltham, MA, USA).  
At least three independent experiments were conducted in triplicate and the results are presented as fold change compared to a control (cells not exposed to cannabinoids), which was set as 1.  
As experimental controls, T (781.2 pM-25.6 \(\upmu\)M) and E\({}_{2}\) (180 fM-367 nM) were tested as positive controls for ER agonism, while Ralooxifene (12.0 pM-24.5 nM; Biosynth Ltd., Berkshire, UK) was used as a positive control for ER antagonism. Regarding AR assays, R1881 (7.8 pM-1 nM) and hydroxyfulamide (OHF; 4.1 nM-9 \(\upmu\)M; Sigma Aldrich Co., St. Louis, MO, USA) were used as positive controls for AR agonism and antagonism, respectively. Stock solutions of all the cannabinoids, as well as T, E\({}_{2}\), Raloifern, R1881, and OHF, were prepared in 100% DMSO and stored at \(-\)20 \({}^{\circ}\)C. Before each experiment, fresh dilutions were prepared in a medium and the final concentration of DMSO in an exposure medium was 0.06%.