DHM (CAS No. 27200-12-0, HPLC ≥ 98%) was obtained from Chengdu Mansite Bio-Technology (Chengdu, China). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO) and palmitate acid (PA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Penicillin/streptomycin and bovine serum albumin (BSA) were obtained from Beyotime (Shanghai, China). Antibodies against acetyl-lysine (AcK), voltage-dependent anion channel (VDAC), P62 and LC3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against beta-actin (ACTB), ATG4B, and SIRT3 were obtained from Abcam (Cambridge, MA, USA). Antibody against optic atrophy 1 protein (OPA1) was purchased from BD Biosciences (San Jose, CA, USA). Chloroquine (CQ), 3-methyladenine (3-MA) and 3-(1H-1, 2, 3-triazol-4-yl) pyridine (3-TYP, CAS No. 120241-79-4) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Other reagents were purchased as indicated in the corresponding methods.
2.2. Cell culture and treatments
HepG2 cell line was provided from the American Type Culture Collection (Manassas, VA, USA). HepG2/ATG4B+/− cell line, in which ATG4B was heterozygous knockout with CRISPR-Cas9 system, was a gift from Nan Zhang (Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038, China) . Both HepG2 and HepG2/ATG4B+/− cell lines were cultured in DMEM medium with 10% FBS, 1% penicillin/streptomycin at 37°C and 5% CO2 incubator, and cells from the 3rd to 6th passages were used for the experiments. To detect the effect of DHM on PA-induced oxidative stress in hepatocytes, both cell lines were pretreated with DHM (20 µM) or the vehicle (0.5% DMSO) for 2 h prior to the treatment of PA (0.2 mM) for an additional 16 h as described previously . To determine the involvement of autophagy and SIRT3 in DHM-induced inhibition of oxidative stress in hepatocytes, the autophagy inhibitor 3-MA (0.5 mM) and CQ (20 µM) as well as a SIRT3 inhibitor 3-TYP (50 µM) were used 1 h prior to DHM treatment, respectively.
2.3. Green fluorescent protein (GFP)-LC3 transfection
After seeded on cell culture dish (NEST, China) for 8 h, HepG2 cells were transfected with GFP-LC3 expression vector (kindly provided by MengYu Liu, Department of Occupational Health, Third Military Medical University, Chongqing 400038, China) at the final concentration of 1 µg/µL for 4 h using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Samples were then analyzed using the laser confocal scanning microscopy (Zeiss, Germany).
2.4. Immunofluorescence microscopy analysis
HepG2 cells were loaded with MitoTracker Red (500 nM, Cell Signaling Technology, USA) to label the mitochondria as described. Briefly, after treated as indicated, the HepG2 cells were loaded with MitoTracker Red and imaged by laser confocal scanning microscopy (Zeiss, Germany) under the excitation (Ex) and emission (Em) wavelength of 644 nm and 665 nm, respectively. To observe the mitochondrial fusion, HepG2 cells were labeled with MitoTracker Red and then fixed with 4% paraformaldehyde (Beyotime, China), permeabilized with 0.25% Triton X-100 (Beyotime, China) and blocked with 5% goat serum (Beyotime, China). Then, cells were incubated with primary antibody against OPA1 at 4°C overnight, and incubated with Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody (1:200, Beyotime, China). Finally, nuclei were counterstained with DAPI. Samples were analyzed using the laser confocal scanning microscopy (Zeiss, Germany).
2.5. Intracellular and mitochondrial ROS assessment
The intracellular and mitochondrial ROS (mtROS) levels were determined using DCFH-DA (Beyotime, China) and MitoSOX™ Red (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, respectively. After treated as indicated, cells were loaded with DCFH-DA (10 µM) in FBS-free DMEM at 37°C for 30 min, then washed twice with FBS-free DMEM to detect the intracellular ROS level. Meanwhile, cells were loaded with MitoSOX™ Red (5 µM) at 37°C for 30 min, then washed gently three times with warm PBS to detect the mtROS level. The mean fluorescence intensity of intracellular ROS (Ex/Em: 488/525 nm) and mtROS (Ex/Em: 510/580 nm) were determined using flow cytometry (FCM; Accuri C6, BD Biosciences, San Jose, CA), respectively.
2.6. MMP measurement
The MMP was measured using JC-1 probe (Beyotime, China, CAS No. 47729-63-5) according to the manufacturer’s instruction. Briefly, after treated as indicated, cells were loaded with JC-1, followed by washing twice in PBS before being subjected to FCM for green fluorescence (Ex/Em: 488/525 nm) and red fluorescence (Ex/Em: 525/590 nm) (Accuri C6, BD Biosciences, San Jose, CA). Fluorescence intensity ratio was calculated to reflect the MMP.
2.7. Transmission electron microscopy (TEM)
The mitochondrial morphology was observed by a TEM. Briefly, HepG2 cells were treated as described previously  and visualized on TEM (EM420, Amsterdam, The Netherlands). Three samples per group and ten images of each sample were selected for quantitative analysis. Any types of ultrastructural changes including broken inner and outer membranes, intramitochondrial edema, disrupted cristae, hypertrophic giant mitochondria as well as intramitochondrial edema were considered damaged and dysfunctional mitochondria. The ratio of the number of normal mitochondria to the total was calculated to evaluate the percentage of normal mitochondria.
2.8. Western blotting and co-immunoprecipitation (Co-IP)
Cells were lysed in cell lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) containing 1% protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). The mitochondrial proteins were extracted using a Cell Mitochondria Isolation Kit (Beyotime, China) with protein concentrations determined using a BCA kit (Beyotime, China), and samples mixed with loading buffer (Beyotime, China) were heated at 100°C for 10 min. For Western blotting, equal amounts of proteins were resolved via SDS-PAGE (10%-15%), followed by transfer and fixation on polyvinylidene difluoride membranes (0.22 µm; Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk for 2 h and then immunoblotted with antibodies (1:1000) overnight at 4°C. Then, the membranes were washed with tris-buffered saline with Tween (TBST) buffer three times, followed by the incubation with the appropriate secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) (1:5000) for 1 h and then washing with TBST buffer three times. The immunostained bands were visualized and determined using Fusion FX (Vilber Lourmat, France) and Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA). ACTB and VDAC were used as loading controls for whole cell lysates and mitochondrial lysates, respectively. Densitometry analysis was carried out using ImageJ software. For Co-IP analysis, the proteins were diluted in lysis buffer and then mixed with the corresponding antibodies as well as protein A/G-agarose bead slurry (Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C with gentle rotation. Then, the mixtures were centrifuged at 1100×g for 3 min at 4°C and washed with lysis buffer five times followed by Western blotting for immunoblotting. The specificity of antibodies used for immunoprecipitation was routinely validated by running negative controls with non-immune IgG under the same conditions.
2.9. Statistical analysis
Data analysis was performed with SPSS 19.0 software (Chicago, IL, USA). All experimental data were expressed as mean ± SD of at least 3 experiments. Statistical differences among groups were determined with either Student’s t-test (for two groups) or one-way analysis of variance (ANOVA) followed by LSD post hoc tests (for multiple group comparisons). A p value less than 0.05 was considered to indicate statistically significant difference; ns means not significant.