Renilla luciferase pgl4.74 hrluc tk plasmid

A 1.5 kb fragment of the proximal IGFBP2 promoter was amplified with the following primer set: IGFBP2-For: 5′-CAGGGTACCCTGTGCCCTTGCTAACCGCCCATTTC-3′, IG FBP2-Rev: 5′-CAGGCTAGCCGGGTCCTAAGGGCCGGCTTCTCC-3′ (restriction enzyme site KpnI and NheI were underlined). The DNA fragment was inserted into the luciferase reporter vector pGL4.18 [luc2P/Neo] (Promega Corporation, Madison, Wisconsin, United States) by KpnI and 3′ NheI (IGFBP2-pGL4.18). For luciferase reporter assay, 5 × 10⁴ HEK293T cells were cultured in 12-well plates overnight. Next day, cells were transfected with 1 μg of with lentiviral empty vector or lenti-RUNX2 together with 20 ng of IGFBP2-pGL4.18 and Renilla luciferase pGL4.74 [hRluc/TK] plasmid (Promega, Madison, WI, United States) using PolyJet^TM In Vitro DNA Transfection Reagent (SignaGen Laboratories, Rockville, MD, United States). At 48 h post transfection, luciferase activity in each well was measured by Dual-Luciferase^® Reporter Assay System (Promega) and normalized to Renilla.

HER2-5/AHRKO cells were transfected with the reporter and expression plasmids and the Renilla luciferase pGL4.74[hRluc/TK] plasmid (Promega; used as an internal standard) using the reverse transfection method with PEI Max^® (Polysciences, Warrington, PA, USA). After transfected HER2-5/AHRKO cells were incubated overnight at 37 °C, the cells were treated with the AHR antagonist StemRegenin 1 (10 μM) for 24 h. Subsequently, the cells were harvested, and luciferase activity was measured using the Dual-Luciferase^® Reporter Assay System (Promega). The activity of firefly luciferase was normalized to that of Renilla luciferase.