Dual light reporter assay system

The plasmid containing Ahr promoter sequence was generated previously [32 (link)]. GCs (10⁵ cells per well) were seeded in serum-free medium on a 24-well plate pre-coated with human fibronectin, followed by transfection with 500 ng of pGL3_Ahr-1792 reporter vector or equimolar amount of empty vector (pGL3-basic) after 1h incubation. 50 ng of pSV-b-galactosidase control vector (pSV-b-gal) (Promega, Madison, WI, USA) was used for evaluating transfection efficiency. Transfections were performed using TurboFect in vitro Transfection Reagent (Thermo Scientific). After 12 h, cells were washed with PBS and new medium containing PMSG or vehicle was added. Reporter gene analysis was performed 12 h later with Dual-Light Reporter Assay System (Applied Biosystems, Bedford, MA, USA) according to the manufacturer’s protocol. GloMax 20/20 luminometer (Promega) was used to detect luciferase and b-gal levels. Results are expressed as luciferase/b-gal expression [relative luciferase units (RLU)] and shown as mean fold changes with respect to control (empty pGL3-basic).

Cells were seeded onto 12-well plates (2 × 10⁵ cells per well) and transfected with the pTN24 splicing reporter plasmid with a constitutively expressed β-galactosidase (β-gal) reporter for transfection normalization and a luciferase (Luc) reporter that was conditional on the excision of a translational stop codon by splicing. The cells were collected 48 h after transfection, and the reporter activity was measured with a Dual-Light Reporter Assay System (Applied Biosystems) and determined by calculating the ratio of Luc to β-gal activity.