Dual glo luciferase assay kit

HepG2 cells were seeded overnight in 24-well plates at a density of 2 × 10⁴ cells/well. The next day, using Lipofectamine 2000 reagent (Invitrogen) and following the manufacturer’s instructions, the cAMP response element (CRE) or FoxO1 firefly luciferase reporter or a mock vector were co-transiently transfected with Renilla expression vector. At 18 h post-transfection, cells were stimulated with C3G (10 and 50 µM) for 24 h. Then, cells were washed with 150 μL phosphate-buffered saline (PBS) per well followed by cell lysis using 40 or 60 μL of Passive Lysis Buffer (PLB) per well (Promega, Madison, WI, USA). Luciferase activities were measured using a Dual-Glo Luciferase Assay kit (Promega). All luciferase emission measurements were quantified using a Victor X2 plate reader (PerkinElmer, Santa Clara, CA, USA). All firefly luminescence signal were normalized to that of Renilla luciferase driven by the constitutively active SV40 promoter (pRL-SV40; Promega).

Amyloidogenic processing of APP was monitored in HEK293 cells essentially as described using an APP-GAL4 reporter assay that drives GAL4 upstream activator sequence (GAL4-UAS)-dependent expression of firefly luciferase [22 (link)]. Briefly, APP fused in frame at its C-terminus to GAL4 was co-transfected into HEK293 cells with a GAL4-UAS-firely luciferase reporter gene (pFR-Luc), Renilla transfection efficiency control plasmid (pRL-CMV) and either KLC1wt or KLC1S460D. BACE1, α-secretase and γ-secretase cleavage of APP releases the APP intracellular domain fused to GAL4 which translocates to the nucleus to regulate expression of firefly luciferase. Transfection efficiency normalised firefly luciferase signals thus provide a readout for amyloidogenic processing of APP. Cells were transfected in 24 well plates at 70–80% confluence with 0.25 μg each of APP-GAL4, pFR-Luc and either KLC1wt or KLC1S460D, along with 0.025 μg of pRL-CMV Renilla control plasmid. Cells were transfected as described above (see Cell culture and transfection). Luciferase signals were developed 24 h later using a Dual-Glo Luciferase assay kit (Promega) according to the manufacturer’s instructions and quantified using a Promega GloMax Navigator luminometer. Full details of the assay including design and construction of plasmids have been described previously [22 (link)].

Cells transfected for 24 hr with Firefly control plasmid and hRluc-GFP-GC-rich or Rluc-GFP-AU-rich plasmids were harvested and processed for RNA (4/5) and protein (1/5) luciferase quantification. Total RNA was purified using Trizol (Invitrogen) and DNAse-treated (Turbo DNAse, Invitrogen). qRT-PCR was carried out as described in the corresponding section, and Renilla mRNA levels were normalized to the Firefly control. Luciferase protein assay was performed with the Dual Glo Luciferase assay kit (Promega) according to the manufacturer’s instructions. Relative light determinations were measured in a Lumat LB 9507 luminometer (Berthold).

Luciferase reporter vectors were constructed as described previously [39 (link)]. Briefly, the constructed wild-type (WT) and mutant (MUT) luciferase reporter vectors were simultaneously delivered into VSMCs. Forty-eight hours post-transfection, luciferase activity was detected using the Dual-Glo luciferase assay kit (Promega, USA).

H1299 cells, seeded in 24-well plates, were transfected with 100 ng of p21-luc, 1 ng p53, 1–100 ng LRH-1 and 10 ng of the renilla luciferase plasmid, pRL-CMV, using FuGENE HD (Promega). Luciferase activities were determined after 24 h, using the Dual-Glo Luciferase Assay kit (Promega). To control for transfection efficiency, firefly luciferase activities were calculated relative to Renilla luciferase activities.

Changes in the activity of IRF1 and STAT1 were assayed using the Cignal Finder multi pathway dual luciferase reporter array (Qiagen Inc., Germantown, MD). Small airway epithelial cells (SAECS) were transfected with luciferase reporter constructs according to the manufacturer’s protocol. SAECS were co-transfected with an inducible IRF1 responsive construct and a constitutively expressing Renilla luciferase construct. The inducible IRF1 and STAT1 responsive construct encodes the luciferase reporter gene under the control of a basal promoter element joined to tandem repeats of an inducible IRF1 and STAT1 dependent promoter sequences. The inducible reporter monitors changes in activity of IRF1 by measuring the increase or decrease of IRF1 and STAT1 dependent luciferase expression. The constitutively expressing Renilla luciferase reporter expression was used to normalize differences in transfection efficiencies between different treatment groups. A constitutively expressing GFP construct was used for visual confirmation of transfection. Transfected cells were stimulated with cytomix ± HTS for 4 hrs. Changes in promoter activity of IRF1 and STAT1 were assayed by measuring the increase or decrease of IRF1 and STAT1 dependent luciferase expression using the Dual Glo luciferase assay kit (Promega Corp., Madison, WI).