Slug promoter

The cDNA encoding N1ICD was subcloned into the expression vector pcDNA3.1(+) between the BamHI and XhoI sites, and subcloning was confirmed with sequencing by Shanghai Genechem Co., Ltd. The pGL3 reporter construct plasmid (−2000/+100) consisted of a 2100-bp genomic DNA fragment of the Slug promoter (Promega, Madison, WI, USA). The pGL3-Slug promoter plasmid or its negative control pGL3-basic plasmid carrying the firefly luciferase reporter were co-transfected with an internal control, pRL-TK Renilla vector (Promega), by using Lipofectamine 2000 (Invitrogen). In addition, cells were respectively transfected with 600 ng of N1ICD overexpression plasmid pcDNA3.1(+) or its negative control pcDNA3.1. Cell lysates were harvested 48 h after transfection. The firefly and renilla activities were measured by the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to the Renilla luciferase activity. Each transfection was repeated three times.