P53 luc

Luciferase reporter constructs including pAP-1-Luc, pCRE-Luc, pGAS-TA-Luc, p-ISRE-TA-Luc, pLILRE-Luc, pNFκB-Luc, p53-Luc, pSTAT3-TA-Luc, and pTARE-Luc were purchased from Agilent Technologies (CA, USA) and Clontech Laboratories, Inc (Japan). HEK293 cells in 100 mm plates at 70–80% confluency were transfected with different reporter plasmids by Lipofectamine 3000 (Thermo Fisher Scientific). Stable transfectants were selected with 75 μg/mL Zeocin for pcDNA3.1-zeo or 400 μg/mL G418 for pcDNA-neo.

The reporter plasmid, p53-Luc, and positive control plasmid pFC-p53 were purchased from Agilent Technologies (Santa Clara, CA). MG-Luc plasmid (kindly provided by Dr. Bert Vogelstein) carrying a mutation in the p53 binding sites was used as a negative control [22 (link)]. WWP-Luc (also provided by Dr. Bert Vogelstein), is a reporter gene with a 2.4-kb p21WAF1 promoter region [23 (link)]. Huh7 cells were transfected with one μg of reporter vectors and 50 ng of pRL-SV40 expressing Renilla luciferase as internal control with DMRIE-C reagent (Invitrogen) according to the manufacturer’s instructions. After 48 h of transfection, luciferase activity was measured according to the manufacturer’s protocol (Promega, Madison, WI). Assays were done in four parallel sets.