β galactosidase plasmid

The mouse Ctss promoter (1425 bp) was amplified by PCR from mouse genomic DNA and cloned into the firefly luciferase reporter plasmid (pGL3-basic, Promega), and co-transfected the renilla luciferase reporter plasmid (pRL-TK, Promega) as internal reference into HEK293 cells (3 × 10⁵ cells per well) with cDNA encoding Hmgb2 (pcDNA3.1FlagmHmgb2, Addgene, plasmid: 31610) or enhanced green fluorescence protein (GFP). The empty vector (pcDNA3.2/V5, 500 ng) was used as a control. Cells were harvested and assayed for firefly and renilla luciferase activities using the dual-luciferase reporter assay system (E1910, Promega) according to the manufacturer's protocol. The data were normalized to the co-transfected β-galactosidase plasmid (Invitrogen) and expressed as the relative luciferase activity (units).

Luciferase reporter plasmid containing the inducible xenobiotic response element (XRE), which directly interact with the aryl hydrocarbon receptor (AhR), was purchased from QIAGEN (Valencia, CA, USA). A reporter plasmid assay was conducted, as described previously [34 (link),35 (link)]. Briefly, HaCaT cells were transiently transfected with an XRE-reporter plasmid and a β-galactosidase plasmid (Invitrogen, Carlsbad, CA, USA), using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Twenty-four hours later, the cells were treated with various concentrations of ACTPER for 6 h. Cell lysates were prepared, and a luciferase activity was measured using the Luciferase Reporter kit (Promega, Madison, WI, USA) with a microplate luminometer (MicroLumatPlus LB96V, Berthold, Germany). Luciferase activity was normalized to β-gal activity.