Renilla reniformis luciferase

Oligonucleotides needed for the production of the luciferase reporter vector were designed based on the expected binding site of the 3′-untranslated region of the STAT3 mRNA. A DNA fragment that was located in the middle of the expected binding site was subjected to PCR amplification, and the resultant product was inserted into the pmiR-RB-ReportTM vector between the Not-I and Xho-I restriction sites (RiboBio Co., Ltd., Guangzhou, China). Oligonucleotides of the wild-type version (wt) and mutated version (mut) of the predicted binding site were constructed (RiboBio Co., Ltd., Guangzhou, China). 293 T cells were cultured in DMEM and cotransfected with 50 ng of empty reporter (vehicle), pmiR-RB-STAT3-wt or pmiR-RB-STAT3-mut, and 50 nM negative control miRNA, miR-106a mimic (no. miR10000385; RiboBio Co., Ltd., Guangzhou, China), or miR-20b mimic (no. miR10003187; RiboBio Co., Ltd., Guangzhou, China) using Lipo6000TM (Beyotime, China). After a waiting period of twenty-four hours, the cells were tested with the Dual-Luciferase Assay System to determine whether or not they contained firefly luciferase activity and Renilla reniformis luciferase activity (Promega, USA).

Gene reporter assays were performed as previously reported [65 (link)]. Cells were transfected with expression vectors (empty vector or FZD7), the human TAZ promoter pGL3-TAZ and renilla reniformis luciferase (Promega, Madison, WI, USA). Cells were then cultured for 24 hours with or without 5 μM ICG-001 or 5 nM Wnt3a. After that time, cells were collected and luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA), according to the manufacturer's instructions.