Cloning and Transfection of pri-miR-181a/b1 Enhancer

The pri-miR-181a/b1 enhancer region was cloned into the pGL4.27 Luc2p/minP vector (Promega) as described^{28 (link)}. Thirty nanograms of pri-miR-181a/b1 Luc2p/Peak 1 was co-transfected with 1 ng Renilla luciferase reporter pRL-TK (Promega) into HEK293T cells, or Luc2p empty vector (negative control). For TCF1 loss-of-function on pri-miR-181a/b1 enhancer activity, TCF7 siRNA (Dharmacon A-019735-13-0005) or non-targeting siRNA (Dharmacon D-001910-01-05) were co-transfected; YY1 siRNA (Dharmacon A-011796-16-0005) was transfected as a positive control. For gain-of-function, different amounts of pCDNA3-HA-TCF7 (0, 30 or 100 ng, Addgene #40620) or pCDNA3-CTNNB1 plasmids (0, 30, 100 ng, Addgene #16828) were co-transfected. As for GSK3β inhibitor treatments, 1 µM BIO or 10 µM SB216763 was added 12 h after transfection. Cells were collected 48 h later, and enhancer activity was determined using the Dual Luciferase Reporter Assay System (Promega, E1910) as described by the manufacturer’s protocol.