Fbs ct

Cancer cell lines were seeded in 24-well plates as follow: 22Rv1, LNCaP, LNCaP-LN3, LNCaP-PRO5 at 1 x 10⁵ cells/well; V16D, MR42D, MR49F, CAMA-1, ZR-75-1, MD-MB-231 and MCF-7 at 5 x 10⁴ cells/well; PC-3, DU-145, SW780, SW1710 and RT4 at 8 x 10⁴ cells/well. Twenty-four hours after seeding, adenoviruses were transduced at a multiplicity of infection (MOI) of 2. Seventy-two hours after infection, cells were lysed, and a luciferase assay was performed as described (Promega, Madison, WI, USA). Protein content was estimated by adding 250 μl of Bradford reagent (ThermoFisher Scientific, Waltham, ON, Canada) to 3 μl of total lysate. Absorbance was then read using an Infinite F50 absorbance microplate reader (TECAN, Mannedorf, Switzerland) at 595 nm. SV40-Luc virus was infected in parallel to normalize for infection efficiency between different cell lines. For androgen sensitivity assessment, cells were treated with 10 nM dihydrotestosterone (DHT) (Toronto Research Chemicals, Toronto, ON, CA) and 10 μM Bica (Sigma-Aldrich, St.Louis, MO, USA) in 10% charcoal-treated FBS (FBS-CT) (Wisent Bio Products), 24 h post-infection. Luciferase assays were performed after 48 h of treatment.

LNCaP, LNCaP-GFP, LAPC4, LAPC9-GFP and 22Rv1 cells (2000 cells/well) were seeded in a 384-well black plate (Greiner Bio-One North America Inc., Monroe, NC, USA) in RPMI-1640 with 10% charcoal-treated FBS (FBS-CT, Wisent) and dihydrotestosterone (DHT) (Toronto Research Chemicals, Toronto, ON, CA) at 1 nM. The cells were then transduced with either CMV-TSTA (1 × 10⁴ infectious viral particle (IVP)) or PSEBC-TSTA (1 × 10⁵ IVP) adenoviruses. Bioluminescent imaging was performed as described below by using LV200 bioluminescence microscope. Media were replaced with appropriate treatments (DHT (1 nM), DHT + 10 μM Enzalutamide (Enza, MedChem Express, Monmouth Junction, NJ, USA), DHT + 10 μM Bicalutamide (Bica, Sigma-Aldrich, Oakville, ON, Canada), DHT + 10 μM Apalutamide (Apa, MedChem Express) DHT + 10 μM Darolutamide (Daro, MedChem Express)). Forty-eight hours later, bioluminescence imaging was repeated on the same cells. D-luciferin (3.5 mM) was added 20 min before each imaging time-point.