Lipofectamine 2000 reagent

HepG2 cells were seeded in 12-well plates at a density of 2×10⁵ cells/well, and when the cells were 50–60% confluent, they were transfected with AMPKα and adiponectin receptor 1/2 siRNA duplex (4 μL/well; Santa Cruz, CA, USA) for 24 h using Lipofectamine 2000 reagent (Promega, Madison, WI, USA). After transfection, the cells were treated with C3G (10 and 50 μM) or A-769662 (100 μM) as the positive control and 0.1% v/v dimethyl sulfoxide (DMSO) as the vehicle for 24 h. The protein levels of AMPKα were analyzed through immunoblotting to confirm knockdown efficiency.

At the beginning, the potential target of miR-317b-5p was predicated by the TargetScan platform (www.targetscan.org), and the SCAI was detected as one of the possible target of miR-317b-5p. The dual-luciferase reporter gene assay was performed to verify the interaction between miR-317b and SCAI. For luciferase reporter assay, the NSCLC cells were transfected with mutant- or wild-type reporter plasmid containing 3′UTR of SCAI to form the reporter vector SCAI Wt type or SCAT Mut type. Then cells were co-transfected with SCAI Wt or SCAT Mut and miR-371b-5p mimic or NC mimic by using Lipofectamine 2000 reagent (Promega, Madison, U.S.A.). After 48-h culturing, the luciferase activities were detected by the dual-luciferase reporter assay system.

The predicted 3′-UTR sequence (295 bp) of HIPK2 was cloned from the genomic DNA of primary hepatocytes, and then the sequence was inserted into the pmir-GLO empty luciferase reporter vector (Promega, WI, USA). The predicted promoter sequence (−300 bp to + 10 bp) of HIPK2 was inserted into the pGL-3 basic luciferase reporter vector from Promega. For further studies, primary hepatocytes were co-transfected with the internal control vector pmir-GLO (Promega) and pmir-GLO-HIPK2 3′-UTR using the Lipofectamine 2000 reagent (Thermo Fisher Scientific) for 20 h, followed by an incubation with the indicated chemicals for 10 h. Similarly, hepatocytes were co-transfected with the empty vector pGL-3 basic (Promega) and the pGL-3 vector containing the HIPK2 promoter using the Lipofectamine 2000 reagent for 20 h; the pRL vector was used as an internal control. Cells were then incubated with the indicated chemicals for 10 h. The luciferase reporter activities were determined using the Dual-Glo luciferase assay system from Promega. The drugs and concentrations used in the present study were as follows: 5 μM resveratrol, 30 μM aspirin, 10 μM vitamin E, and 15 μM ursolic acid. The compounds were purchased from MedChemExpress NJ, USA).