Pgl3 her3 luc

The promoter regions of HER2 and HER3 genes were cloned into the pGL3-Basic luciferase vector (Promega Corp., Madison, WI) to produce the luciferase reporter plasmids pGL3-HER2-Luc and pGL3-HER3-Luc. All of the constructs were verified by Sanger sequencing. The primers for plasmid constructs were presented in Supplementary Table S3. Next, to test promoter activity of HER2 and HER3 genes regulated by EHF, FTC133 cells were transfected with pcDNA3.1(−)A-EHF or empty vector in 6-well plates and were cotransfected with pGL3-HER2-Luc, pGL3-HER3-Luc, and pRL-TK plasmids (Promega Corp., Madison, WI) using Lipofectamine 3000 (Invitrogen, Grand Island, NY). The pRL-TK plasmid, containing Renilla luciferase, was commonly used to normalize transfection efficiency. Cells were harvested 36 h post-transfection, and luciferase activities were analyzed on EnSpire^® Multimode Plate Reader (PerkinElmer, Waltham, MA) using the dual-luciferase reporter assay system (Promega Corp., Madison, WI) according to the manufacturer's instructions. Data were expressed as relative luciferase activity (Firefly luciferase activity/Renilla luciferase activity). Each experiment was performed in triplicate.

To construct luciferase reporter plasmids, the promoter regions of HER2-4 genes were amplified from genomic DNA of BGC823 cells. The amplification products were digested with restriction enzymes and inserted into pre-digested pGL3-Basic luciferase vector (Promega Corp., Madison, WI, USA) to produce the luciferase reporter plasmids pGL3-HER2-Luc, pGL3-HER3-Luc and pGL3-HER4-Luc. All of the constructs were verified by Sanger sequencing. The primers for plasmid constructs were presented in Supplementary Table 5.
To test the promoter activity of HER2-4 genes modulated by EHF, BGC823 or 293T cells were transfected with pcDNA3.1(-)A-EHF or empty vector in six-well plates and were cotransfected with pGL3-HER2-Luc, pGL3-HER3-Luc or pGL3-HER4-Luc, and pRL-TK plasmids (Promega Corp., Madison, WI, USA) using Lipofectamine 3000 (Invitrogen, Grand Island, NY, USA). The pRL-TK plasmid, containing Renilla luciferase, was used to normalize transfection efficiency. Cells were collected 36 h post-transfection, and luciferase activities were analyzed on EnSpire Multimode Plate Reader (PerkinElmer, Waltham, MA, USA) using the dual-luciferase reporter assay system (Promega Corp., Madison, WI, USA) according to the manufacturer's instructions. Data were expressed as relative luciferase activity (Firefly luciferase activity/Renilla luciferase activity). Each experiment was performed in triplicate.