Prl tk luc renilla luciferase

The BEAS-2B cells were seeded at 1 × 10⁵ cells/well in a 96-well plate in K-SFM complete media. At 80% to 90% confluency, cells were transfected with pRL-TK-Luc Renilla luciferase and pGL4.43[luc2P/XRE/Hygro] (purchased from Promega, Madison, WI, USA) plasmids at a 1:10 ratio using FuGENE HD transfection reagent (Roche Applied Science, Indianapolis, IN, USA). For ELK-1 reporter assay, pELK1-Luc (Signosis, Santa Clara, CA, USA) plasmids were transfected together with pRL-TK-Luc Renilla plasmid. After 24 h post-transfection, media was replaced by supplement-free K-SFM, and cells were co-treated with carvedilol and 10 µM B(a)P for 24 h. Cells were lysed with passive lysis buffer (Promega, Madison, WI, USA), and the firefly luciferase activity was measured using a dual luciferase reporter assay kit (Promega, Madison, WI, USA). Measurements were performed using a single mode luminometer (GloMax® 20/20 Luminometer, Promega, Madison, WI, USA). The ratio of firefly luciferase to Renilla luciferase was normalized to the negative control for AhR/XRE or ELK-1 promoter activity.

Dual luciferase assay for NF-κB has been described in previous work.²¹ In brief, HEK-293 cells were transfected with pRL-TK-Luc Renilla luciferase (Promega, Madison, WI) and pGL4.22-NF-κB (Promega) plasmids at a ratio of 1:30 using FuGENE HD Transfection Reagent (Roche Applied Science, Indianapolis, IN). Twenty-four hours later the cells were exposed to TNF-α (10 ng/ml) and incubated in fresh growth medium containing drugs for an additional 5 hours. Cell lysates were analyzed by the dual luciferase reporter gene assay (Promega) with Renilla luciferase serving as a normalization factor.