Dual luciferase reporter assay system

The impact of ENL on NF-κB activity was assessed via a NF-κB Cignal Reporter Assay (Qiagen) and quantitative RT-PCR for NF-κB target gene expression. For the NF-κB Reporter Assay, cells were seeded at a density of 2.5 × 10³ in 96-well plates. After 24 h, the cells were transfected with the NF-κB Reporter mixture (an inducible NF-κB-responsive firefly luciferase reporter + a constitutively expressing Renilla construct) using FuGENE^Ⓡ 6 (Promega, Madison, WI, USA). After another 24-h incubation, cells were treated with vehicle, 1 µM or 10 µM ENL in complete media for 48 h, followed by the same treatments plus LPS (10 ng/ml) in complete media for 24 h. The cells’ luciferase activity was measured using Promega’s Dual Luciferase^Ⓡ Reporter Assay System on a Cytation 3 Cell Imaging Multi-Mode Reader (BioTek Instruments Inc.). For NF-κB target gene measurement, cells were seeded at a density of 1.5 × 10⁵ in 6-well plates. After 24 h, cells were treated with vehicle, 1 µM or 10 µM ENL in complete media for 48 h, followed by the same treatments plus LPS (10 ng/ml) in complete media for 24 h. Four NF-κB target genes were chosen to assess in vitro from the 41 target genes with significantly lower expression in SDG mice versus controls; criteria for inclusion were a known link to breast cancer progression and P < 0.01 for the in vivo NF-κB target gene array.

In order to validate DMS measurements, we characterized a series of individual variants using both a fluorescence-based reporter (22 (link)) and a dual-luciferase reporter for −1PRF in the SINV 6K gene. Fluorescence reporters bearing polyprotein variants of interest were transiently expressed in HEK293T cells, and the relative mKate: eGFP intensity ratios were determined by flow cytometry as previously described (22 (link)). HEK293T cells were transiently transfected with dual-luciferase reporter constructs using lipofectamine 3000 (Invitrogen, Carslbad, CA) in accordance with the manufacturer's instructions. Cells were harvested two days post-transfection and lysed using the Passive Lysis Buffer provided in the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). This lysis buffer contains CHAPS detergent that should solubilize any remaining uncleaved, membrane bound reporter enzymes. The relative activities of the firefly luciferase and renilla luciferase domains in the clarified lysate were then measured using the Dual-Luciferase Reporter Assay System on a Synergy Neo2 plate reader (Biotek, Winooski, VT) in accordance with the manufacturer's instructions. To determine the −1PRF efficiency, the firefly: renilla luciferase ratio was normalized relative to that of a construct lacking the insert as was previously described (34 (link)).