BMP2 Promoter Luciferase Assay in BMSCs

The pGL4 luciferase vector harboring human BMP2 promotor sequence was constructed by GenePharma (Shanghai, China). The Renilla luciferase vector was obtained from Cell Signaling Technology (Albany, NY, USA). Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) reagent was used for transfection according to the manufacturers’ instruction. The human bone marrow mesenchymal stem cells (BMSCs) were isolated and kept in our laboratory as previously described [20 (link)]. In general, the human BMSCs were seeded into a 12-well plate and grown until 70–80% confluence. For transfection, 1 μg plasmid DNA with 1 μL Lipofectamine^TM 3000 were mixed in 500 μL Opti-MEM^TM (Thermo scientific, Waltham, MA, USA), and added into the culture medium. After 12-h incubation, cells were treated with 0.5 μM sesamin (59867, Sigma-Aldrich, St. Louis, MO, USA), 100 nM estradiol (E8875, Sigma-Aldrich, St. Louis, MO, USA), 0.2 nM amcenestrant (Sigma, St. Louis, MO, USA) or the combinations for an additional 24 h. For dual-luciferase assay, Dual-Luciferase^® Reporter Assay Kit (Promega, Madison, WI, USA) was applied as mentioned before [20 (link)]. The luciferase activity was measured by PerkinElmer VictorTM X2 2030 multilateral reader (PerkinElmer, Waltham, MA, USA). The firefly luciferase activity was normalized to renilla luciferase activity.