Pgl4.43 luc2p xre hygro

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.

pGL4.43[luc2P/XRE/Hygro] (Promega) was used to establish HT-29-AhR and Caco-2-AhR reporter cell-lines by electroporation using the Nucleofector® device (Lonza) according to the manufacturer’s recommendations. Stable AhR reporter cell lines were selected using Hygromycin (600 μg/mL for HT-29 and 200 µg/mL for the Caco-2 cell line, InvivoGen) and validated using TCDD at 10 nM final concentration.