Dual luciferase assay system

A luciferase construct containing wild-type (WT) and mutated (MUT) binding site vectors of LINC01679 3′ -untranslated region (3′UTR) or WT and MUT binding site vectors of SLC17A9 3′UTR (Promega, Madison, Wisconsin, United States) was co-transfected with scramble or miR-3150a-3p mimic/inhibitor into cells grown within 24-well plates by the use of Lipofectamine 3000 (Thermo Fisher Scientific, Inc). Later, we used a dual-luciferase assay system (Promega) to analyze luciferase activities of transfected cells in accordance with specific instructions.
A luciferase construct containing WT and MUT binding site vectors of SLC17A9 3′UTR (Promega, Madison, Wisconsin, United States) was co-transfected with NC/vector or LINC01679-KD/LINC01679-OE into cells grown within 24-well plates by the use of Lipofectamine 3000 (Thermo Fisher Scientific, Inc). Later, we used a dual-luciferase assay system (Promega) to analyze luciferase activities of transfected cells in accordance with specific instructions.

HeLa cells were transiently transfected with 100 ng HES1-Luc reporter, 10 ng pCS2-N1ICD, 100 ng pCS2-MAML1, and 20ng pRL-TK in a 96-well plate. The HES1-Luciferase reporter has been described previously [24 (link)]. Cells were harvested after 40–48 h and the levels of luciferase were measured with the Dual Luciferase Assay System from Promega on a GloMax-96 or GloMax-Multi+ Luminometer from Promega (Madison, WI, USA). The error bars on graphs represent standard deviation of three independent experiments.
HeLa cells were transiently transfected with 100 ng HES1-Luc reporter, 10 ng pCS2-Notch1 ICD, 100ng FLAG-MAML1 or FLAG-MAML1K/R and 20 ng pRL-TK in a 96-well plate. Cells were harvested after 40–48 h and the levels of luciferase were measured with the Dual Luciferase Assay System from Promega on a GloMax-96 or GloMax-Multi+ Luminometer from Promega (Madison, WI, USA). The error bars on graphs represent standard deviation of three independent experiments.
HeLa cells were cotransfected with 100 ng pG5-luc reporter and 40 ng GAL4-N1 ICD, 100 ng p300-HA, 100 ng CDK8-FLAG and 150 ng MAML1 plasmids using TransIT-LT1 transfection reagent (Mirus, Madison, WI, USA). After 48 h, the cells were harvested and luciferase activity was measured using LucySoft3 (Anthos Labtec, Salzburg, Austria). The bars represent standard deviations of three replicate samples.

2 × 10⁵ RAW264.7 cells were cotransfected with 50 ng AP-1 reporter construct, 400 ng pcDNA-vector, -DUSP12, and -DUSP12-PM together with 10 ng of pRL-null plasmid using Lipofectamine LTX reagent (Invitrogen). Equal amounts of pcDNA3.1 empty vector were transfected as control. Reporter activity was determined with Promega Dual Luciferase Assay System (Promega). Firefly luciferase values were normalized for transfection efficiency by means of the Renilla luciferase activity that is constitutively expressed by pRL-null.

The pGL3 plasmid carrying the CCL3 promoter region (from -2000 base pairs to the transcription start site (TSS)) and the luciferase reporter gene were constructed by YouBio (Changsha, China). The pRL-TK plasmid which carried renilla luciferase and pGL3 plasmid carried the CCL3 promoter region were co-transfected into THP-1 cells at approximately 70% confluence using MegTran (T210003, Origene, USA). After 12 h, Fludarabine (5 μM), which is the STAT1 phosphorylation inhibitor, or 2-NP (45 μM), which is used to enhance STAT1 transcription, were added separately to the cells. Luciferase activity was assessed according to the standard protocol of the Promega Dual Luciferase Assay System (E1910, Promega, USA) after 24 h stimulation. Luciferase activity values were normalized by the corresponding renilla luciferase activity.

HEK293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and a 100 U/ml penicillin-streptomycin mixed solution. Cells were seeded into 35 mm-diameter plates and transfected with the expression plasmids and TP53 reporter plasmids using polyethylenimine MW 25000 (Polysciences, Warrington, PA, USA). After 24 h, firefly and Renilla luciferase activities were determined with the Promega Dual Luciferase Assay System (Promega, Madison, WI, USA). The pRL-EF vector, which expresses Renilla luciferase under the control of the EF-1α promoter, was used to normalize the transfection efficiency of the luciferase reporters. The values shown are the averages of experiments repeated three times, with each transfection performed in duplicate for each experiment.