Cpt1α

For western blotting, 10–30 μg of whole-cell extract, cytoplasmic protein, or mitochondrial extract were separated by 10% SDS-PAGE. Blots were probed with antibodies diluted 1:1000 in 5% skim milk against MATα1 (ab129176), PIN1 (07-091, Millipore), TOM20 (ab78547), DDK (TA50011-100, Origene), His-Tag (A00186-100, GenScript), OXPHOS (ab110413), CK2α (2656, Cell Signaling), CPT1α (12252, Cell Signaling), SDHα (5839, Cell Signaling), MCAD (ab110296), JNK (9252, Cell Signaling), pJNK (4668, Cell Signaling), p38 (9212, Cell Signaling), p-p38 (9211, Cell Signaling), GSK3β (9315, Cell Signaling), p-GSK3β (9336, Cell Signaling), methyl-lysine (ab23366), α-Tubulin (32-250022125, Invitrogen), β-actin (A3854 Sigma), and GAPDH (5174 Cell signaling Technology). HRP-linked secondary antibodies anti-rabbit (7074S Cell Signaling) and anti-mouse (7076S Cell Signaling) were diluted 1:5000 in 5% skim milk.

Fetal bovine serum (FBS), culture media, TRIzol reagent, LysoTracker Red, BODIPY493/503, and the Lipofectamine 3000 transfection reagent were obtained from Invitrogen (Carlsbad, CA, USA). Palmitate (PA) and oleic acid (OA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against LC3, SQSTM1/p62, and TFE3 were obtained from Sigma-Aldrich. Antibodies against Cathepsin L, PGC1α, PPARα, CPT1α, and ACOX1 were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against VPS11 and β-Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Unless otherwise specified, all other reagents were purchased from Sigma-Aldrich.

The HepG2 cells were collected and washed twice with PBS; then, the cells were lysed in RadioImmunoprecipitation Assay (RIPA) buffer containing 100 mmol L⁻¹ phenylmethylsulfonyl fluoride and 25 mmol L⁻¹ proteinase inhibitor (Roche, Switzerland). The total protein concentration was assessed using a BCA Protein Assay Kit (Beyotime Biotechnology, China). Approximately 40 μg of sample protein was separated using 10% Mini-RROTEAN TGX SDS-Stain-Free Precast gels (Bio-Rad, USA) and then transferred to polyvinylidene fluoride membranes (Millipore, USA). After transfer, the membrane was blocked with 5% non-fat dry milk at room temperature. Specific primary antibodies were incubated with the blot at 4 °C overnight. The signal was detected with HRP-conjugated anti-IgG followed by detection with ECL (Millipore) and the images were scanned using a ChemiDoc Touch Imaging System (Bio-Rad). Antibodies against STAT3, phospho-STAT3, IL-6, TNF-α, G6pase, PEPCK, IRS2, Glut2, FAS, CPT-1α, IRS-1, phospho-IRS-1(Ser307), AKT, phospho-AKT (Ser473), FOXO1, phospho-FOXO1 (Ser256), Caspase3 and β-actin were purchased from Cell Signaling Technology (USA). Antibodies against SREBP-1C and ACCα were purchased from Santa Cruz Biotechnology (USA). Antibodies against Bax and Bcl2 were purchased from Abcam (UK).