Renilla luciferase reporter plasmid

MCF-7 and MDA-MB-231 cell lines were transiently co-transfected with the 489 bp BiP promoter luciferase reporter construct and a Renilla luciferase reporter plasmid (Promega) as an internal control using Lipofectamine 3000 (Invitrogen). COS-1 cells were transiently co-transfected with a BiP promoter luciferase reporter construct, Myc-CEMIP or empty vector as a control, and the Renilla luciferase reporter plasmid. DNA was incubated with polyethyleneimine (MW = 250,000 kDa, Polysciences, Inc.) for 30 minutes at room temperature before addition to the cells, and the medium was changed 18 hours later. 48 hours post-transfection, cells were lysed using 1X passive lysis buffer, and luciferase assays using the Dual-Glo Luciferase Assay System (Promega) were performed as recommended by the manufacturer. Luminescence was recorded using a SpectraMax L microplate reader (Molecular Devices). BiP promoter induction of firefly luciferase was normalized to the Renilla luciferase signal.

CHO-K1 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM: 5% FBS, 2 mM L-glutamine, 1 mM L-proline, 10 mM HEPES) in humidified 5% CO₂ atmosphere at 37°C. For Wild-type (WT) reporter construct, 228bp of 3′UTR of Stard13 gene spanning putative site for mmu-miR-125a-5p was cloned downstream of firefly luciferase coding region in pMIR-REPORT vector (Ambion, Austin, TX). To generate the mutated construct (MUT), site-directed mutagenesis of the putative target was carried out in the WT-3′ UTR construct. Primers used for the cloning and mutagenesis are listed in the Table 2. CHO-K1 cells were plated in 24 well plate one day before the transfection. Cells were co-transfected with 100ng of WT-3′UTR or MUT-3′UTR firefly luciferase reporter construct, 0.5 ng of renilla luciferase reporter plasmid (Promega, Madison, WI) and with either miR-125a-5p mimic(GE lifesciences) or Negative control (NC) mimic (Ambion catalog# 4464058) in duplicates. Cell lysates were collected 48hours after transfection and assayed for luciferase activity using Dual-Luciferase Reporter Assay System (Promega) and Victor 3 Multilabel Counter 1420 (PerkinElmer). Renilla luciferase activity was used to normalize the firefly luciferase activity.

EBs were transfected on day 0 using polyethyleneimine (PEI, Sigma-Aldrich, 408727). The following plasmid DNAs were used (all at a concentration of 0.8 μg/well of a 12-well plate), either separately or by cotransfection, if desired: plasmid expressing nondegradable β-catenin mutated on S33 (Professor H.R. Korswagen, Utrecht University, Netherlands), plasmid overexpressing mouse Hif1α (Professor Poelinger Lab, Karolinska Institutet, Sweden), and empty pcDNA3 plasmid for control transfections. Luciferase reporter assay was used to quantify the β-catenin-mediated transcriptional activation of TCF/LEF binding sites in 2-day-old EBs. Aggregates cultivated in agar microwells (12-well plate) were transfected on day 0 with 0.8 μg of Super8XTOPflash/Super8XFOPflash luciferase plasmid (Professor R.T. Moon, University of Washington, USA) together with 0.4 μg Renilla luciferase reporter plasmid (Promega, Madison, WI, USA). 48 hours after transfection, reporter activity was measured using the Dual-Luciferase Reporter Assay System Kit (Promega). Analyses of each experimental condition were performed in triplicates. The luciferase activity of SuperTOP/FOPflash was normalized to the Renilla luciferase signal.

Cells were co-transfected with a pGL4.18-SKP2 promoter reporter plasmid^{58 (link)} and a control Renilla luciferase reporter plasmid (Promega, Madison, WI, USA) for 24 h. At the indicated time, the cells were treated with simvastatin 20 μg/ml for 24 h and then harvested. Luciferase activity was determined using a dual-luciferase reporter assay system (Promega). Light units were normalized to Renilla luciferase activity.