Pgl4.36 luc2p mmtv hygro vector

CV-1 cells were seeded into 24-well plates and cultured for 24 h before transfection. Prior to transfection, the medium was replaced with 10% charcoal dextran-treated FBS–DMEM. After 4 h, a DNA mixture containing an MMTV-luciferase reporter plasmid (pGL4.36[luc2P/MMTV/Hygro] vector, 0.3 μg, Promega) with (TGTTCT)₆ as the enhancer element sequence, and an internal control plasmid pRL-SV-40 (5 ng), was transfected using the TransFast reagent (Promega, Madison, WI, USA). After transfection, cells were treated with 10 μg/mL compounds (samples) or 1 μM spironolactone. After 2 h, the treated cells were further treated with 1 nM dexamethasone and incubated for an additional 24 h. The luciferase activities of the cell lysates were measured using the Dual-Luciferase Reporter Assay System, according to the manufacturer’s instructions (Promega). The relative luciferase activity was normalized to the corresponding Renilla luciferase activity to determine transfection efficiency.

HeLa cells were grown in a 96-well white plate (1 × 10⁴ cells/well) in DMEM containing 5% FBS without phenol red for 24 h and transfected with the pGL4.36 [luc2P/MMTV/hygro] vector or the pGL4.32 [luc2P/NF-κB-RE/hygro] vector from Promega (Madison, WI, USA). Cells were treated with analytes, as indicated in the figure legends, after which luciferase assays were performed according to manufacturer’s protocol.

LNCaP.FGC (LNCaP) cells (RCB2144) were purchased from Riken Cell Bank (Ibaraki, Japan). pGL4.36 [luc2P/MMTV/Hygro] vector was purchased from Promega (Madison, WI, USA). The murine mammary tumor virus long-terminal repeat sequence function was used as androgen and glucocorticoid sensors [28 (link)]. Stably overexpressing LNCaP cells were generated by transfecting the reporter vector with polyethyleneimine. Clones were selected using 100 µg/mL hygromycin and isolated independently, and three clones were obtained. Reporter activity was evaluated using the One-Glo Luciferase Assay System (Promega). The luminescence activity was measured using a GloMax^® Discover Microplate Reader (Promega). However, the following points should be noted with caution: within 24 h of seeding, cells in the exponential growth phase showed almost no response to testosterone or DHT; therefore, treatment was initiated after incubation of ≥48 h or more.
For cell viability testing, Cell Counting Reagent SF (Nacalai Tesque) was used according to the manufacturer’s protocol as previously described [29 (link)]. Briefly, confluent cells in 96-well plates were treated with reagents for 24 h, then 10 μL of WST-8 reagent was added to each well. After 90 min, cell viability was determined by measuring the absorbance at 450 nm.