TCF Activity Modulation by DKK3 Transfection

In order to confirm TCF activity change by DKK3 transfection, a TOP/FOP Flash assay was performed as previously described13 (link). HSC-3 cells were seeded into 24-well plates at 1.0 × 10⁵ cells/well and cultured for 24 h. Cells were then cotransfected with 0.2 μg of DNA constructs, 0.2 μg of reporter plasmid, and 0.2 μg of internal control pRG-TK, then cultured for a further 24 h. Luciferase activity was measured and normalized for transfection efficiency using a Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA). Graphs show the average of three independent experiments with normalized transfection efficiency using Renilla luciferase. As a positive control, cells were stimulated by 100 ng/ml of rhEGF (R&D) for 24 h14 (link). To confirm the TCF reporter assay, one more experiment was done using Cignal™ Reporter Assay Kits (QIAGEN). As a control, cells were stimulated by 50 mM LiCl for 24 h.