Dual-luciferase reporter assay

The USF1 binding sites on the ATF2 gene promoter were predicted by UCSC (http://genome.ucsc.edu/) and JASPAR (http://jaspar.genereg.net/) websites. The oe-NC and oe-USF1 were respectively co-transfected with ATF2-2 Kb luciferase reporter plasmid or the recombinant luciferase reporter plasmid with the truncated binding site. 48 h after transfection, cells were harvested and lysed. The luciferase activity was detected using a luciferase assay kit (K801-200, BioVision, Milpitas, CA, USA) and analyzed in a dual luciferase reporter gene system (Promega, Madison, WI, USA). Renilla luciferase was used as an internal reference, and the degree of activation of the target reporter gene was compared based on the ratio of the relative luciferase units of firefly luciferase divided by the relative luciferase units of Renilla luciferase.