β gal enzyme assay system

Cells were transfected with 5 μg of M50 Super 8x TOPFlash plasmid (a kind gift from Dr. Randall Moon [20 (link)] ) or 5 μg of β-gal by using Dharmafect Transfection Reagent. At 24 h after transfection, cells were harvested by using the Luciferase Report Assay System (Promega, US) or the β-gal Enzyme Assay System (Promega) according to manufacturer’s instructions. β-catenin activity was calculated by the TOPflash after normalization of β-gal.

The promoter sequence of AGT (−306/+36) was retrieved as previously described^{16 (link)}. The putative STAT3 binding sites in the promoter sequence of AGT were determined using the PROMO bioinformatics web server^{17 (link)}. The PCR products of the AGT promoter were generated using cDNA from BEAS-2B cells. The AGT promoter construct was cloned by ligating the PCR products to the pGL3-basic vector at the Nhel/HindIII restriction sites. Mutations were introduced at the STAT3 binding site of the AGT promoter using a QuikChange II Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA).
Activity of the AGT promoter was measured with a reporter gene assay performed using a luciferase assay system (Promega Corp., Madison, WI, USA) according to the manufacturer’s protocol. Briefly, the cells were transfected with the luciferase vector containing the promoter sequence of AGT (pGL3-AGT) or the empty vector (pGL3) along with pSV-β-galactosidase. The cells were subsequently harvested with passive lysis buffer, and the luciferase activity was monitored using a microplate luminometer (Berthold Technologies GmbH & Co. KG, Germany). β-gal was used as the control for normalizing the transfection efficiency, and its activity was measured using a β-gal enzyme assay system (Promega).