For transcription factor plasmids, the ORFs (open reading frames) of sterol regulatory element-binding protein 1 (SREBP1, XM_021624594.1), sterol regulatory element-binding protein 2 (SREBP2, XM_021625095.1), HNF1α (XM_021621309.1), CCAAT-enhancer-binding protein α (CEBPα, NM_001172496.1), CEBPβ (NM_001124447.1), CREB1 (XM_021597386.1), FOXO1 (XM_021618954.1), RXRα (XM_021590263.1), P65 (XM_021578194.1), and peroxisome proliferator-activated receptor γ (PPARγ, XM_021610844.1) were cloned into PCS2+ plasmids (Invitrogen, Carlsbad, CA, USA) by the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China).
HEK 293T cells were cultured according to the method described in the previous studies [12 (link)]. Transfection was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the instructions. A total of 200 ng of promoter reporter plasmid, 600 ng of transcription factor plasmid, 20 ng of pRL-TK renilla luciferase, and 2.0 μL Lipofectamine 3000 were co-transfected in the 24-well plate in triplicate wells with three independent experiments. The TransDetect Double-Luciferase Reporter Assay Kit (TransGen, Beijing, China) and InfiniTE200 plate reader (Tecan, Männedorf, Switzerland) were used for the detection of dual luciferase activity.
HEK 293T cells were cultured according to the method described in the previous studies [12 (link)]. Transfection was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the instructions. A total of 200 ng of promoter reporter plasmid, 600 ng of transcription factor plasmid, 20 ng of pRL-TK renilla luciferase, and 2.0 μL Lipofectamine 3000 were co-transfected in the 24-well plate in triplicate wells with three independent experiments. The TransDetect Double-Luciferase Reporter Assay Kit (TransGen, Beijing, China) and InfiniTE200 plate reader (Tecan, Männedorf, Switzerland) were used for the detection of dual luciferase activity.
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