Dual glotm luciferase assay

Human hepatocellular adenoma HepG2 cells were maintained in D-MEM with 5% FBS. Cells were added to 96-well plates and grown to approximately 80% confluence. Plasmid DNA including pGL3 reporter vectors, the pRL-CMV luciferase (as control for transfection efficiency), pCDNA3-HNF4α2 (Addgene), pCMV-CCAAT/enhancer-binding protein α (C/EBPα) (gift from Dr. Magnus Nord, Karolinska Institute), or pCDNA3 were complexed with Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA) and applied to individual wells, according to the manufacturer’s protocol. Transfected cells were lysed with passive lysis buffer (Promega) 24 h after transfection. Promoter activities of cell lysates were quantified by Dual-Glo^TM luciferase assay (Promega) with the control values of pGL3-Basic vs pRL-CMV set at 1.0. To study the role of SP1 in mediating the transactivation of human miR-194-2 proximal promoter by HNF4α, the SP1 inhibitor mithramycin was added 1 h after transfection and cells were lysed 24 h after transfection for dual-luciferase assay.

HepG2 human hepatocellular adenoma cells were maintained in D-MEM with 5% FBS. Cells were added to 96-well plates and grown to approximately 80% confluence. Plasmid DNA including pmiR-Chd1 (or pmiR-H3f3), the pRL-CMV luciferase, and a synthetic mimic of miR-194/miR-192 (miScript miR-194/miR-192, QIAGEN Inc, Valencia, CA), or AllStars Negative Control siRNA (QIAGEN, as negative control for microRNAs) were co-transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol of DNA-RNAi co-transfection. Transfected cells were lysed with passive lysis buffer (Promega) 24 h after transfection. Promoter activities of cell lysates were quantified by Dual-Glo^TM luciferase assay (Promega) with the control values of pmiR-Chd1/pmiR-H3f3 vs pRL-CMV set at 1.0.
Animal care and use statement: The animal protocol was designed to minimize the pain or distress to the mice. Age-matched young-adult HNF4α Liv-KO mice and their wild-type control littermates were fed rodent chow (#8064, Teklad; Harlan, Indianapolis, IN). Mice were housed at an ambient temperature of 22 °C with alternating 12-h light/dark cycles and allowed water and feed ad libitum.