Pgl3 plasmid

Wild-type and mutant UHRF1-promoters were cloned downstream of the Renilla reporter gene into the PGL-3 plasmid (GenePharma) and transfected into YAP1 overexpressing 293T cells and the relevant controls, using Lipofectamine 3000 (Thermo Fisher Scientific). 3′UTR site-specific mutagenesis at the predicted sites for each target was performed using the QuikChange Site-Directed Mutagenesis Kit, as described by the manufacturer (Agilent, Beijing, China). Cells were harvested 48 h after transfection and analyzed with a Dual-Luciferase Reporter Assay Kit (GeneCopoeia, MD, USA), according to the manufacturer's protocol.

The SIRT6 reporter, which contains 1 kb of the SIRT6 promoter, was created using RT-qPCR as aforementioned and cloned into the pGL3 plasmid (Shanghai GenePharma Co., Ltd). The wild type (WT) and mutant type (MUT) of SIRT6 promoter were co-transfected with Ov-KLF5, Ov-NC, and pRL-TK (internal reference plasmids expressing renilla luciferase) into PDLSCs. The cells were lysed according to the protocol of TransDetect Double-Luciferase Reporter Assay Kit (FR201-01, TransGen Biotech, Beijing, China) following 48 h of transfection, and the supernatant was then collected. The luciferase activity was detected by Dual-luciferase reporter assay system (E1910, Promega). Firefly luciferase/renilla luciferase was adopted as the relative luciferase activity [20 (link)].