Measurement of AhR activation via AhR gene luciferase activity assay

For determination of AhR activation, NHEKs were transfected by using the FugGENE HD transfection reagent (Promega, Madison, WI, USA) with the pGudLuc6.1 reporter plasmid (generously gifted by M. Denison, U.C. Davis) (300 ng/well) in which the firefly luciferase gene expression is dependent on AhR activation. The co-transfected pGL4.74 renilla luciferase plasmid [hRluc/TK] (Promega, Madison, WI, USA) (30 ng/well) functioned as reference plasmid to control transfection efficiency. Medium was changed 6 h after transfection. One day after transfection, cells were stimulated with the AD cytokine mixture (IL-4, IL-13, TNFα and IL-22; each 10 ng/mL) with the plant extract or vehicle for 24 h as described above. Luciferase activity was determined by following the protocol of firefly/renilla Dual Luciferase activity assay (Sigma-Aldrich, St Louis, MO, USA). AhR activation was calculated as the ratio of firefly to renilla luciferase activities in each sample.