4.4. Luciferase Reporter Assays

HBx expression vector (genotype D), pCI-HBx or pCI-GST-HBx was constructed by cloning GST-HBx or HBx from a pGEX-HBx vector into pCI-neo (Promega, Tokyo, Japan). HeLa cells were seeded on 6-well plate at a density of 4×10⁵ cells/well. After 24 h, the cells were co-transfected with 0.2 mg of pCI-neo, pCI-HBx, or pCI-GST-HBx, and 0.19 mg of PathDetect Cis-Reporting Systems pAP-1-Luc, or pNF-κB-Luc (Agilent Technologies, Tokyo, Japan) as well as 0.01 mg of pGL4.74 [hRluc/TK] (Promega) for expression control using Effectene transfection reagent (Qiagen, Tokyo, Japan) according to the instructions. Luciferase assay was performed 48 h after transfection using PicaGene Dual Sea Pansy Luminescence Kit (Toyobo, Osaka, Japan) and measured by Lumistar Optima (BMG labtec, Saitama, Japan) according to the instructions. HepG2 cells were seeded on 24-well plate. After 24 h, the cells were transfected with 0.25 mg of pCI-neo or pCI-HBx, 0.25 mg of Fluc vector, and 0.01 mg of Rluc vector using FuGENE HD Transfection Reagent (Promega) according to the instructions. The compound was added 24 h after the transfection and the luciferase assay was performed 72 h after that.