Luciferase reporter assay system

Plasmids or chemically synthesized small interfering RNAs (siRNAs) (siSTAT1, genOFF h-STAT1_1999A_SIGS0000954-1; siTOP2A, si-h-TOP2A_101; standard, 5nmol_ siG000007153A-1-5; siTOP2B, genOFF st-h-TOP2B_001, 5nmol_stB0004038A-1-5; RiboBio, Guangzhou, China) were transfected into 293T cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Cells were collected and lysed with passive lysis buffer (Promega). The luciferase activity of cell extracts was measured through the luciferase reporter assay system (Perkin Elmer, United Kingdom) according to the manufacturer’s instructions.

For plasmid transfection, cells were transfected with the indicated plasmids using FuGENE-HD (Promega Biosciences) according to the manufacturer’s instructions. The transfection was carried out at 40% cell density. For 12-well plates in transfection experiments, 1 μg of pDNA and 2 μl of FuGENE-HD were used.
For RNA interference, FlexiTube siRNA targeting NRF2 (SI03246614 [#1, Target sequence is 3’ UTR region.], SI03246950 [#2] and SI4223009 [#3]) (QIAGEN) was transfected into cells at 20 nM using Lipofectamine RNAiMAX (Invitrogen). A control siRNA (ALLStars negative control siRNA, 1027281, QIAGEN) with no known mammalian homology was used as a negative control. The transfection was also performed at 40% cell density. For 12-well plates in transfection experiments, 2 μl of siRNA (final concentration of 20 nM) and 4 μl of Lipofectamine RNAiMAX were used.
Luciferase activity of transfected cells was measured using a luciferase reporter assay system (Perkin Elmer) and Fluoroskan Ascent FL (Thermo Scientific, Lafayette, CO, USA). The luciferase activity was normalized to co-transfected GFP fluorescence.