Nanodlr stop glo reagent

TERT promoter regions were cloned into a pNL1.1 vector (Promega), transformed in TOP10 chemically competent cells (Lifetech), and verified by Sanger sequencing. To generate methylated versus unmethylated plasmids, 1 μg of plasmid was treated with SssI (NEB) or mock as per manufacturer’s instructions. To ensure complete methylation, plasmid DNA was treated with SssI twice. Cells in culture were seeded into 96 well plates and transfected in suspension using X-tremeGENE HP DNA Transfection Reagent (Roche). Transfection complexes were prepared such that each reaction contained 11 μl Opti-MEM, 59 ng empty pCR2.1-TOPO vector, 5 ng pGL4.53(luc2/PGK) vector (Promega), 1 ng pNL plasmid, and 0.12 μl HP transfection reagent. After 2 days, transfected cells were lysed in 50 ul passive lysis buffer (Promega), transferred to black 96 well plates, and measured for reporter activity by mixing 50 μl of ONE-Glo EX Luciferase Reagent and NanoDLR Stop & Glo Reagent sequentially as described in the Nano-Glo Dual Luciferase Assay System manual (Promega).

HEK293T cells were plated at 10,000 cells per well in nine six-well plates in 100 μl of growth media and then cultured for 24 h at 37°C. Each well was then transfected with a mixture of 80 ng of plasmid (42 ng of carrier plasmid (E488B, Promega), 35 ng firefly plasmid, and 3 ng of reporter plasmid), and 0.24 μl of transfection reagent (FuGENE 6, Promega). After culture for 21 h, 50 μl of media was aspirated from each well, and 50 μl of firefly substrate solution (ONE-Glo Ex Reagent, Promega) was added to each well. Samples were incubated for 30 min at 23°C. A 50-μl aliquot of the nanoluciferase substrate solution (NanoDLR Stop & Glo Reagent, Promega) was then added to each well. Samples were incubated for another 30 min at 23°C. Firefly and nanoluciferase luminescence were measured using standard luminescence protocols (Clariostar Microplate Reader, BMG Labtech).

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.