2.1 Human sample and TMA assay

Tumor specimens for tissue microarrays were obtained from 354 HCC patients who underwent surgical procedures between April 2005 and September 2008 at the Department of Liver Surgery, Zhongshan Hospital of Fudan University. All patients were duly informed about the purpose of the study and consented to the use of their samples. Tissue microarray (TMA) was conducted as previously described [12, 13]. Evaluation of immunohistochemical staining was also carried out as previously described [14]. All images were analyzed using a computer-automated method (Image-pro plus 6.0, Media Cybernetics, Silver Springs, MD, USA) [15].

2.2 Cell culture and animals model

HCC cell lines L-02, MHCC97-L, MHCC97-H, HCCLM3, SNU-449, and Huh7 were from Song Shushu (Zhongshan Hospital, Fudan University). L-02, MHCC97-L, MHCC97-H, HCCLM3, and Huh7 was cultured in DMEM medium supplemented with 10% FBS, 100 unit/ml streptomycin, 100 μg/ml penicillin. SNU-449 was cultured in RMPI-1640 medium supplemented with 10% FBS, 100 unit/ml streptomycin, 100 μg/ml penicillin. and all cell lines were incubated at 37 °C in a humidified atmosphere with 5% CO2. All cell culture reagents were obtained from Gibco (Invitrogen, USA).

male BALB/c nude mice weighed 18–20 g and aged 4–6 weeks were purchased from the Shanghai Model Organisms Center, Inc. (Shanghai, China). All model mice were maintained in specific pathogen-free conditions. Humane care of animals was objected to the “Guide for the Care and Use of Laboratory Animals” criteria of the National Academy of Science (National Institute of Health publication 86–23, revised 1985).

Limiting dilution Xenograft arrays evaluate cancer cells’ tumorigenic capacity, making them useful for studying cancer stem cells. Huh7 cells are serially diluted (from 107 to 105 cells) and implanted into nude mice to evaluate tumor formation.

For the mouse model of liver metastasis, each mouse was primed with 200 µL of SNU-449 cell suspension by tail intravenous injection. Two weeks later, intraperitoneal injections of MK-2206 (40 μg/g of body weight) or DMSO were given to all the mice twice a week for a total of two weeks. After that, they were killed, and the liver tissue was removed and preserved in a paraformaldehyde solution to create tissue slices.

2.3 Quantitative real-time PCR (qRT-PCR)

Total cellular RNA extraction was performed using a RNeasy mini kit (Qiagen, Germany) and cDNA was synthesized using the Quantitect Reverse Transcription Kit (Qiagen, Germany) according to the manufacturer’s instructions. Target genes were quantified using FastStart Universal SYBR Green Master (Roche diagnostics, Germany) and DNA amplification was carried out using a LightCycler 480 (Roche Diagnostics, Germany). The relative quantities of target gene mRNAs compared to an internal control were determined using the ΔCq method. PCR conditions were as follows: 5 min at 95 °C, followed by 40 cycles of 95 °C for 10 s and 60 °C for 60 s. GAPDH was used as an internal control. Primers and probes are listed in Table S1.

2.4 Western blot analysis

Protein extraction from the tissue or cell samples was performed using radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease and phosphatase inhibitor cocktail. The protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit.

Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 hour at room temperature to prevent non-specific binding.

The membranes were then incubated overnight at 4 °C with primary antibodies specific to the target proteins. After washing with TBST, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. The blots were washed again with TBST and the protein bands were visualized using an enhanced chemiluminescence (ECL) detection system.

The intensity of the protein bands was quantified using image analysis software and normalized to the intensity of the loading control, typically beta-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

The antibodies used in this study are listed in Supplementary Table S5.

2.5 Colony formation assay

Once they reached a diameter of 100 μm, HCC spheres were collected through gentle centrifugation, dissociated with trypsin-EDTA (Invitrogen, USA), and mechanically disrupted with a pipette. The resulting cells were gently centrifuged to remove trypsin. Single cells were seeded in DMEM with 10% FBS (Gibco, USA) at a density of 2000 cells per well in a 6-well plate (Corning, USA). Parental Huh7 cells were seeded at the same density as a control population to evaluate colony-forming capacity. After two weeks, the colony-forming ability was assessed by counting the number of colonies (> 70 cells) under a microscope after staining with crystal violet (Sigma-Aldrich, USA). Representative images were photographed using an Olympus LX-71 fluorescence microscope. Experiments were performed in triplicate.

2.6 Sphere-forming assay

Serum-free medium for sphere culture was composed of DMEM/F12 medium supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, 20 ng/ml human recombinant epidermal growth factor, 20 ng/ml human recombinant basic fibroblast growth factor, 1% nonessential amino acids, 1% GlutaMax, 2% B27 supplement (Invitrogen, USA), and 1% methylcellulose (Sigma, USA). HCC cells were cultured at a density of 1000 cells/ml; when spheres reached a diameter of 100 μm, the sphere-forming efficiency was calculated and spheres were collected for further use.

2.7 Transwell assay

Transwell assays were used to assess migration activity. The cells were collected and washed with 1x PBS. To conduct migration tests, 5 × 104 cells were seeded in an upper chamber with a non-coated membrane (24-well insert, pore size 8 μm; Corning, USA) in DMEM containing 1% FBS. The lower chambers contained DMEM with 10% FBS as a chemoattractant. Cells were incubated at 37 °C for 24 h. Cells that had migrated or penetrated the bottom surface of the membrane were preserved with 4% methanol and then stained with crystal violet. Stained cells were counted in ten randomly selected 100X microscopic areas. All experiments were conducted in triplicate. The unpaired two-tail student’s t-test was used for comparison.

2.8 Phosphorylation kinase array

The Proteome Profiler Human Phospho-Kinase Array Kit (R&D Systems) was used to simultaneously assess the phosphorylation status of various kinases. Cell lysates were prepared post-stimulation and their protein concentrations determined using a BCA protein assay kit. The array membranes were blocked with array buffer 1 for 1 h, then incubated with 300 μg of cell lysates overnight at 4 °C. After washing, the membranes were further incubated with a cocktail of biotinylated detection antibodies, followed by a horseradish peroxidase-conjugated streptavidin incubation. Protein spots were visualized using a chemiluminescent detection reagent, and signal intensities were quantified using a digital imaging system and image analysis software. The relative phosphorylation levels of the kinases were determined by comparing the signal intensities of individual kinases with the control. Experiments were performed in triplicate, with data expressed as mean ± standard deviation (SD). Statistical significance was determined using a two-tailed t-test, with a p-value of less than 0.05 considered statistically significant.

2.9 Immunoprecipitation (IP)

Cells were transfected as mentioned and collected using immunoprecipitation lysis buffer (Beyotime, Shanghai, China). Equal volumes of cell lysis were then treated for 6 h at 4 °C with moderate rotation with FLAG antibody (1:200; Sigma) immobilized onto Protein G-Sepharose beads. The beads were afterwards washed with lysis buffer three times. Following addition of SDS-PAGE sample loading buffer and subsequent boiling, the beads were centrifuged to obtain supernatant for western blot.

2.10 Nucleoplasmic separation arrays

The Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) was used to collect and lyse cells in accordance with the manufacturer’s instructions. Using a different lysis buffer and centrifugation, the nuclear and cytoplasmic components of the lysate were separated. Western blot was utilized to analyze the proteins present in the arrays.

2.11 Statistical analysis

Differences between the two groups were analyzed using a two-tailed t-test, while a two-way analysis of variance (ANOVA) was employed to evaluate the statistical significance of two-factor interactions across multiple time points. The X-tile software was used to determine the optimal cut-off point for continuous variables. The cut-off point was subsequently used to divide HCC patients into groups of high and low GPX8 expression. The survival analysis was conducted using the Kaplan-Meier method with a log-rank test to assess risk factors for overall survival (OS), disease free survival (DFS) and recurrence free survival (RFS) in HCC patients. Furthermore, a multivariate Cox regression analysis was performed to identify predictors of prognosis. All data were analyzed using the SPSS software (version 24.0, SPSS Inc.). Statistical significance was considered when the P-value was below 0.05 (P < 0.05).