Renilla luciferase reporter gene assay system

Cells were seeded in a 24-well plate and transfected with the luciferase reporter plasmids, pGL4-SP-B (−3000/+450) or pGL4-SP-C (−3000/+120), pcDNA3-Foxp2, pcDNA3-CtBP2, and pcDNA3-MCRIP1 as indicated, together with the Renilla luciferase plasmid as an internal control. The cells were assayed for luciferase activity 48 h after transfection, using Twinlite Firefly and Renilla Luciferase Reporter Gene Assay System (Perkin Elmer).

The BRE assay described in Mallet et al.^{16 (link)} has been modified to assess the functionality of the 5’UTR ENG variants. Briefly, NIH-3T3 were seeded in 96-wells white plates (15,000 cells/well) in DMEM containing 1% fetal calf serum and transfected the following day by a mixture of plasmid (i) BRE luciferase reporter plasmid (75 ng) (ii) pRL-TK luc encoding Renilla luciferase (20 ng), pcDNA3-ALK1 (0.15 ng) and pcDNA3.1-L-ENG WT or 5’UTR-mutated constructs (0–10 ng/well). Four hours after transfection, cells were stimulated overnight with 5 pg/ml of BMP9 in serum-free medium (R&D Systems) and luciferase activity was measured with the twinlite Firefly and Renilla Luciferase Reporter Gene Assay System (PerkinElmer). Means of triplicate were calculated for each sample and Firefly/Renilla ratio of stimulated wells was normalized to that obtained in non-stimulated wells.