Prl sv40 renilla luciferase reporter plasmid

Cells were seeded overnight in 65 µL complete medium in white flat bottom 96-well tissue culture plates (PerkinElmer, Waltham, MA, USA) at 15,000 to 35,000 cells per well to attain 70–90% confluency at the time of transfection. Firefly luciferase reporter plasmids and Rlip-LNA or scrambled control were transfected in 10 µL each of Opti-MEM reduced serum medium (Thermo Fisher) using Lipofectamine 3000 with p3000. The pRL-SV40 Renilla luciferase reporter plasmid (Promega) served as an internal standard and was transfected simultaneously with the firefly luciferase reporter plasmids. Owing to the considerable difference in signal intensity between the various reporter constructs and between unmethylated reporters and their methylated counterparts, all wells transfected with different constructs or different treatments were separated by one blank well to reduce the effects of light bleed on wells of lower signal intensity. Firefly and Renilla luciferase signals were analyzed on a SpectraMax ID3 microplate reader (Molecular Devices, San Jose, CA, USA) using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions. The responsiveness to RLIP knockdown by locked nucleic acid (Rlip-LNA, 50–100 ng/well) relative to scrambled control was evaluated for both unmethylated (UM) and in vitro methylated (M) variants of each reporter at 24 h.

The dual firefly and Renilla reporter assay was performed to test HMOX1 antioxidant response element (ARE)-dependent transcriptional control. Briefly, cells were seeded into 12-well plates at a density of 2 × 10⁵ per well and incubated overnight. Cells were then transiently co-transfected with HMOX-1 ARE-FLuc reporter plasmid (from Reen Wu, UC Davis, CA, USA) or firefly luciferase empty vector, as well as pRL-SV40 Renilla luciferase reporter plasmid (Promega, Madison, WI, USA) (serving as an internal control). At 48 h post-transfection, cells were transferred to Arg-free medium for an additional 24 h prior to dual luciferase assay. HMOX-1 ARE-FLuc firefly and Renilla luciferase activities were assessed by adding ONE-Glo™ EX Reagent and NanoDLR™ Stop & Glo® Reagent sequentially using Dual-Luciferase^® Reporter Assay System according to manufacturer’s instruction (Promega). Firefly luminescence and Renilla luminescence were measured by SYNERGY H1 microplate reader. All the firefly luminescence readings were normalized with Renilla luminescence values and presented as the relative fold differences compared to the control group.