Luciferase reporter assay system

The mouse Akt3 gene promoter region containing CpG island 146 (CpG146) was generated by PCR using mouse genomic DNA. The 5′ primer, bearing a SacI site, was 5′-GCCTCGCCCAGGTGAATGT-3′, and the 3′ primer, bearing a NheI site, was 5′-CGCCAGCAGCGACAGCATCA-3′. The Akt3 promoter fragment was inserted into the pGL3-Basic vector, which includes luciferase as a reporter gene. For the luciferase assay, cells were washed with phosphate buffered saline and harvested with luciferase cell culture lysis reagent. Akt3 promoter activity in the cells was measured with the Luciferase Reporter Assay System using a Sirius luminometer (Titertek-Berthold). Luciferase activity was calculated in relative light units and normalized to the pCMV-RL vector containing the Renilla luciferase as control reporter.

HEK293 cells (8 × 10⁴) were cultured in 500 µl DMEM including 10% FBS overnight using a 24 well plate at 37 °C, 5% CO₂. 1 µg of each plasmid construct was transfected into HEK293 cells using Lipofectamine® 2000 (Thermo Fisher Scientific). A Renilla luciferase plasmid, pCMV-RL (Liu et al., 2018 (link)), was co-transfected with each construct for normalization. After a further 24-hour incubation, cells were washed with phosphate buffered saline and harvested with luciferase cell culture lysis reagent from Dual-Luciferase® Reporter assay kit (Promega). Bcl-2 promoter activity in the cells was measured with the Luciferase Reporter Assay System using a Sirius luminometer (Titertek-Berthold, Bad Wildbad, Germany). Luciferase activity was calculated in relative light units and normalized to the pCMV-RL vector containing the Renilla luciferase as control reporter.