2.5. AP-1 Luciferase Reporter Assay

The Dual-Luciferase reporter assay was performed as described previously [28]. Briefly, the ESCC cells were seeded in a 24-well plate and transfected with pGL3-AP-1 (#40342, Addgene) or pGL3-Basic control vector along with the renilla luciferase reporter construct pRL-SV40for 24 h. Cells were then treated with isoliquiritigenin for another 24 h and harvested for firefly luciferase and renilla luciferase activity determination using the Dual-Luciferase reporter assay system (#E1910; Promega, Madison, WI, USA). Renilla luciferase activity was used as a control for transfection efficiency.