Oleic Acid-Induced Autophagy in HepG2 Cells

The human hepatoblastoma cell line HepG2 was grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA). Oleic acid (Sigma-Aldrich, St. Louis, MO) was conjugated to albumin, and cells were treated with 400 μM Oleic acid for 24 h. BafA1 (50 nM, Sigma Inc., St. Louis, MO, USA) was used to inhibit the autophagy flux. PI3K inhibitor LY294002 (50 μM) (Cell Signaling Technology, Beverly, MA) was used to inhibit autophagy by pre-treating cells for 5 h. HepG2 cells were transfected with Fugene HD (Promega, Madison, WI) for the transfection of plasmids encoding GFP-LC3 or with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) for the transfection of DRAM siRNA and control siRNA (the sequences of the siRNA were obtained from ref.^{38 (link)}), VPS34 siRNA (ACGGTGATGAATCATCTCCAA), ATG5 siRNA (GAAGUUUGUCCUUCUGCU), or PINK1 siRNA (CCTCGTTATGAAGAACTAT) (the sequences of the siRNA were obtained from refs.^{60 (link),61 (link)}. Cells grown on glass cover slips were used for immunofluorescence detection.