Measuring Gli Promoter Activity in DPSCs

To test Gli promoter activity, reporter assay was performed with Gli-responsive luciferase reporter construct (8×Gli-Luc) (gift from Dr. Fernandez-Zapico^{48 (link)}). DPSCs (1×10⁶) were transfected with 3 μg 8×Gli-Luc and 0.6 µg pRL-TK Renilla luciferase (Promega, internal control) with Fugene HD transfection reagent. Cells were induced with OS medium for 3 days and then stimulated with 1 μg/mL of recombinant mouse Shh N-terminus (R&D Systems, Minneapolis, MN, USA) for 8 h. Cells were harvested and colored by the Dual-luciferase Assay Kit (Promega, Madison, WI, USA) and the relative luciferase activity was measured with the Veritas microplate luminometer (Turner Biosystem, Sunnyvale, CA, USA). The experiment was conducted in triplicate.