Vdac1

Protein homogenates were made from the left kidney or the cultured TKPTS cells. Western blot analyses were conducted following the manufacturer’s instructions with specific primary antibodies: fibronectin (15613-1-AP, Proteintech, Rosemont, IL, USA), College 1 (14695-1-AP, Proteintech), α-SMA (ab5694, Abcam), tyrosine hydroxylase (TH, ab112, Abcam), p53 (A3185, ABclonal Technology, China), p21 (14-6715-81, Invitrogen), γ-H2AX (#9718, Cell Signaling Technology, Danvers, MA, USA), α_2A-AR (DF3076, Affinity Biosciences), β-arrestin2 (10171-1-AP, Proteintech), VDAC1 (55259-1-AP, Proteintech), COX IV (11242-1-AP, Proteintech), and NF-κB p65 (sc-8008, Santa Cruz Biotechnology). Anti-GAPDH (AC001, ABclonal Technology) or β-actin antibody (AC026, ABclonal Technology) was adopted for loading controls on membranes. Subsequently, the horseradish peroxidase (HRP)-conjugated secondary antibodies (Servicebio, China) were applied to combine the primary ones for 90 min at room temperature. Finally, the ECL system was used to visualize proteins, and the Image J software (NIH, USA) was applied to analyze bands.

For immunofluorescence analysis, liver samples or AML12 cells were processed and images were obtained as previously described (Liu et al., 2018 (link)). In brief, samples were fixed with 4% paraformaldehyde, immersed with 0.2% Triton X-100 and blocked with 10% goat albumin. They were then incubated with primary antibody(VDAC1 at 1:500 dilution, Proteintech), (Parkin at 1:300 dilution, Abcam, ab15494), LC3B (at 1:200 dilution, CST), followed by incubation with IgG-FITC-conjugated second antibody (at 1:500 dilution, Beyotime technology). Nucleus were stained with 2.5 g/ml DAPI (Sigma-Aldrich).To detect the colocalization of LC3 and a mitochondria marker (MitoTracker Red, Thermo Scientific.USA) in AML12 cells, mitochondria were prestained with MitoTracker Red (50 nM) for 30 min. Besides, we used Ad-mCherry-GFP-LC3B to monitor autophagy flux. In brief, AML12 cells were seeded (1 105 cells/well) in a 24-well-plate and infected with Ad-mCherry-GFP-LC3B (40 MOI) for 24 h and then cultured in fresh medium for another 24 h. Afterwards, the cells were treated with or without PA/OA (200 μM), Corilagin (20 μM), 3-MA (5 mM), and rapamycin (50 nM). Images were captured using the Leica MZ10 F modular stereomicroscope (Leica).

For validation, we employed the identical samples that had been utilized in the mass spectrometry analysis. BCA protein assays were used to determine protein concentration. For each sample, proteins of 30 μg were electrophoresed with 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Membranes were blocked with 5% TBST containing skimmed milk powder for 1 h at room temperature, and incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: β-actin (1:2000,20536–1-AP, Proteintech, China), MFN2(1:2000,12186-1-AP, Proteintech, China), GRP75 (1:2000,14887-1-AP, Proteintech, China),VDAC1(1:2000,155259-1-AP, Proteintech, China), IP3R(1:500,19962-1-AP, Proteintech, China), CytC (1:2000,10993-1-AP, Proteintech, China), TOM20 (1:3000,11802-1-AP, Proteintech, China), COXIV(1:6000,11242-1-AP, Proteintech, China), BNIP3(1:1000,WL01139,Wanleibio, China), PI3K (1:500,WK02240,Proteintech, China), p-PI3K(1:1000,#AF3242, Affinity, China),AKT(1:5000,10176-2-AP,Proteintech, China), p-AKT(1:5000,66444-1-Ig,Proteintech, China). After incubation with antibody diluent containing secondary antibody for 1 h at room temperature, membranes were scanned on ECL detection system (chemiluminescence). Data from densitometry of protein band intensities was analyzed with ImageJ.

The HCC cell lines LM3, Huh-7, BEL-7402, Hep3B, SMMC-7721 and normal liver cells LO2 were purchased from the Chinese Academy of Sciences Committee Type Culture Collection Cell Bank (Shanghai, China). The LO2 cells were cultured in RPMI-1640 with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin, and the HCC cells lines were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM, Logan, UT, USA) at 37°C in a humidified atmosphere of 5% CO₂.
MJ, 5-Fu, adriamycin, 2-DG, dimethyl sulfoxide (DMSO) and glucose-6-phosphate (G-6-P) were purchased from Sigma Aldrich (St. Louis, MO, USA) and stored at −20°C for future use. Z-VAD-FMK and sorafenib were obtained from Selleck Chemicals (Shanghai, China). The final concentrations of DMSO did not exceed 0.1%. All antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The Annexin V/PI apoptosis detection kit was purchased from BD Biosciences (San Jose, CA, USA), the co-immunoprecipitation (IP) kit was from Thermo Fisher Scientific (Waltham, MA, USA) and the antibodies (HK2 and VDAC1) used in IP were from Proteintech Group (Chicago, IL, USA).

Paraffin-embedded tissue specimens were sectioned, deparaffinized in xylene and rehydrated with graded ethanol solution. Antigenic retrieval was then processed with sodium citrate. IHC staining was performed using Streptavidin Peroxidase IHC assay kit (ZSGB-bio, China). The antibodies against KMT2A (Novus Biologicals, dilution 1:50), VDAC1 (Proteintech, dilution 1:200) were used.

All primary antibodies utilized in this study included Bax (1:1000), Bcl2 (1:1000), Bad (1:1000), Caspase-3 (1:1000), Cyt C (1:1500), Cleaved-Caspase9 (1:1500), Cleaved-Caspase-3 (1:1000, all from Cell Signaling Technology, USA), p-p53 (1:1000), p53 (1:1000, Abcam, UK), PEDV N (1:1000), GAPDH (1:5000), β-actin (1:5000), and VDAC-1 (1:3000, ProteinTech Group, Wuhan, China). Secondary antibodies were peroxidase-conjugated secondary antibodies (1:1500, ProteinTech Group, Wuhan, China).

Antibodies used in this paper: RIPK1(Proteintech,17519-1-AP), P-RIPK1(S166) (Proteintech,28252-1-AP), MLKL (CST,14993), P-MLKL (S358) (CST,91689), VDAC1 (Proteintech,55259-1-AP), β-Actin-HRP (MBL, PM053-7). The experimental protocol of sample extraction and electrophoresis was described elsewhere [26 ].