Luciferase reporter assays

We seeded 1 × 10⁵ 293T cells into 24-well plates before transfection. Cells were transfected with the TET3S and SMCHD1 expression vectors, 47.5 ng of pGL3 luciferase reporter vector (methylated or unmethylated), and 2.5 ng of internal control Renilla luciferase reporter vector (pRL-CMV, Promega, Madison, WI). We harvested the cells 48 hours after transfection. All transfections were carried out at least in three independent experiments and in triplicate. Firefly and Renilla luciferase activities were assayed with the Dual-Luciferase assay kit (Promega) according to the manufacturer’s instructions. The firefly luciferase activities were normalized relative to Renilla activity.