Transfection and luciferase reporter assay

GCK overexpression plasmids or GCK 3′-UTR luciferase reporter plasmids were transfected into 293T cells in the presence of different treatment. For each transfection, 2.0 μg of plasmid was mixed with 100 μl of Dulbecco's modified Eagle's medium (without serum and antibiotics) and 4.0 μl of Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA). The mixture was incubated at room temperature for 20 min and added to 12-well plates containing cells and complete medium. The cells were incubated for 30 h and harvested using reporter lysis buffer (Promega, San Luis Obispo, CA, USA) for determination of luciferase activity. Cells were co-transfected with a β-galactosidase reporter plasmid to normalize experiments for transfection efficiency.