Dual-luciferase reporter assay

As per the manufacturer's instructions, Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used to co-transfect cells cultured in 24-well plates with siRNA, expression plasmids pGL3 or pGL3 containing the Claudin-4 promoter and a plasmid that expressed Renilla luciferase. After 24 h, the cells were collected, lysed and imported into a dual-luciferase reporter assay system (cat. no. E1910; Promega Corporation) to determine the luciferase activity. The results were normalized by comparing the relative firefly luciferase activity with the Renilla luciferase activity.