Dual-luciferase reporter assay

GC-2 cells (4.0 × 10⁵ cells/well) were seeded onto 24-well plates and incubated at 37 °C for 24 h. For repressive activity of Zfp819-KRAB, when the cells reached approximately 80–90% confluence, they were co-transfected with 250 ng of pcDNA3.1/myc-Zfp819-KRAB, 250 ng of a firefly luciferase-encoding vector (pGL3-promoter, Invitrogen), and 5 ng of pRL-TK (Renilla used as an internal control for normalization of transfection efficiency in the absence and presence of Zfp819 overexpression). After 24 h, the cells were lysed with passive lysis buffer (Promega), dual luciferase assays were performed with a Luciferase Reporter Assay kit (Promega), and the luciferase activity was measured using a Centro LB 960 DLReady microplate illuminometer (Berthold Technologies). GAL4-DBD was used as a basic control, and KOX1-DBD was used as a positive control. For promoter activity of Zfp819, Tnrc6b or Anxa11 promoter regions were inserted into pGL-promoter (Invitrogen). When the cells reached approximately 80–90% confluence, they were co-transfected with 250 ng of pcDNA3.1/myc-Zfp819 or pcDNA3.1/myc, 250 ng of a firefly luciferase-encoding vector (pGL3-promoter, Invitrogen), and 5 ng of pRL-TK (Renilla). Each experiment was repeated three independent times in triplicate.