Luciferase reporter assay for β-catenin transcriptional activity

The β-catenin luciferase reporter plasmid (pBARL) was obtained from Dr. Randall Moon (University of Washington, Seattle, WA, USA). This plasmid consists of 12 TCF response elements (or β-catenin binding sites) cloned upstream of Promega’s minP© minimal promoter [19]. MC3T3-E1 cells and calvarial osteoblasts were transfected in suspension with the β-catenin luciferase reporter vector using FuGENE 6 or X-tremeGENE 9 according to manufacturers’ instructions. Cells were subsequently plated at a density of 3.0 × 10⁴ cells/cm² on Falcon 48-well plates (or Nunc 48-well plates for calvarial cells) in culture medium. At 1 day post-transfection, cells were placed in serum-free medium and incubated overnight. On the day of the experiment, cells were treated with test substances and subsequently incubated for 24 h. Cell lysates were then prepared by incubation with 65 μl (or 100 μl for calvarial cells) of passive lysis buffer, 1× per well at room temperature for a minimum of 30 min with agitation. To assess luminescence, 15 μl of lysate was combined with 15 μl of Bright-Glo Luciferase Reagent in a 96-well white plate (Greiner Bio-One, Monroe, NC, USA). Reactions for each sample were performed in triplicate. Luminescence was measured using 2-s integration per well on a LMAX II³⁸⁴ microplate reader (Molecular Devices, Downingtown, PA, USA).