Prl sv40 renilla vector

The pGL3 basic vector (Promega, Madison, WI, USA) and the pRL-SV40 Renilla vector (Promega, Madison, WI, USA) were used in all luciferase assays. The pGL3 vector containing the EVI1 promoter was a gift from K. Mitani, Dokkyo Medical University School of Medicine, Kitakobayashi, Japan. Different G2DHE variants were cloned into this vector using the restriction enzymes BamHI and SalI (Table S1). The pGL3 vector containing the EVI1 promoter and full-length G2DHE was described previously [16 (link)]. Site-directed mutagenesis was carried out using the QuikChangeII XL kit (Agilent Technologies, Santa Clara, CA, USA) (Table S2). Mutated constructs were re-cloned into a fresh backbone vector. Cells were seeded at a density of 0.5 × 10⁶ cells/mL and transiently transfected with X-tremeGENE HP DNA (Roche, Basel, Switzerland) transfection reagent according to the manufacturer’s protocol. Per 0.5 × 10⁶ cells 100 ng of pRL-SV40 and 900 ng of the full-length G2DHE constructs were used. Equimolar amounts were used for shorter pGL3 constructs. Cells were harvested 48 h after transfection. Dual-luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocol on a Victor X3 plate reader (Perkin Elmer, Waltham, MA, USA). Luciferase signal was normalized to Renilla signal.

The LINC00842 gene promoter sequence containing YY1 binding site (−2000 bp to transcription start site) or its mutated forms was cloned into the pGL4.10 vector (Promega). The constructs were co-transfected with the pRL-SV40 Renilla vector (Promega) into PDAC cells. The luciferase activity was determined 48 h after transfection by a Dual-Luciferase Reporter Assay System (Promega) and normalized using Renilla luciferase activity.