图1 ANAPC10 在HCC中的表达及与生存预后的关系Note： A/B. Comparison of ANAPC10 mRNA levels between HCC tumor tissues (tumor) and adjacent non-tumor tissues (non-tumor) in the TCGA_LIHC (A) and CHCC_HBV (B) datasets, analyzed by Student's t test. C. Comparison of ANAPC10 protein expression levels between HCC tumor tissues (Tumor) and adjacent non-tumor tissues (Non-Tumor) in the CHCC_HBV dataset, analyzed using Student's t-test. D/E. Kaplan-Meier survival analysis of overall survival (OS) stratified by high versus low ANAPC10 mRNA expression levels in the TCGA_LIHC (D) and CHCC_HBV (E) cohorts. F. Kaplan-Meier survival analysis of OS based on high versus low ANAPC10 protein expression levels in the CHCC_HBV cohort. G. t-SNE clustering plot of hepatocytes from different tissue sources (Normal, Tumor, Lymph, and PVTT) in the GEO single-cell RNA-seq dataset GSE149614. H. t-SNE clustering map displaying ANAPC10 expression levels in hepatocytes from HCC samples in the GSE149614 single-cell dataset. I. t-SNE clustering of different cell types (B cells, Endothelial cells, Fibroblasts, Hepatocytes, Myeloid cells, and T/NK cells) in HCC samples from the GSE149614 single-cell dataset. J. t-SNE clustering visualization of ANAPC10 expression across all cell populations in HCC samples from the GSE149614 dataset. K.Bubble chart illustrating ANAPC10 expression levels categorized by cell type (B cells, Endothelial cells, Fibroblasts, Hepatocytes, Myeloid cells, and T/NK cells) in the GSE149614 dataset.

Fig 1 Expression of ANAPC10 in hepatocellular carcinoma and its association with patient survival

图2 ANAPC10 敲减肿瘤细胞的增殖情况及 Anapc10 敲除小鼠HCC的形成情况Note： A. qPCR was used to analyze the mRNA expression levels of ANAPC10 in various hepatocellular carcinoma cell lines (HepG2, Huh7, MHCC-97H, PLC/PRF/5, SNU-398, HCCLM3) relative to the normal hepatocyte cell line LO2. B. WB was employed to validate the protein levels of ANAPC10 after stable knockdown using shRNA in SNU-398 and MHCC-97H cell lines. C. A CCK-8 proliferation assay [with (n=6) replicates] was carried out in SNU-398 and MHCC-97H cell lines after stable ANAPC10 knockdown. Absorbance was measured at 450 nm (λ=450). D. An immunocompetent C57BL/6 mouse orthotopic HCC model was established by hydrodynamic tail-vein injection of plasmid solutions (2 mL in saline). The control group received a combination of 12 μg pT3-c-myc, 10 μg pX330-p53, 6.5 μg CMV-SB13, and 12 μg lentiCRISPR-v2. The knockout group received 12 μg pT3-c-myc, 10 μg pX330-p53, 6.5 μg CMV-SB13, and 12 μg sgAnapc10. E. Representative macroscopic images of livers with orthotopic HCC tumors were taken 34 d post-injection (n=6 per group, Ctrl vs sgAnapc10). Scale bar=10 mm. F. Hepatic tumor burden was quantified (n=6 per group, sgCtrl vs sgAnapc10). Tumor burden was calculated as the mean of (tumor area / liver area×100%). Statistical analysis was performed using Student's t-test. G. WB was used to validate the knockout efficiency of Anapc10 in tumor tissues (n=3 per group, sgCtrl vs sgAnapc10). Proteins were extracted from samples pulverized in liquid nitrogen. H. Representative H-E and Ki67 immunohistochemical staining of tumor tissues were conducted (scale bar=50 μm). Quantitative analysis of Ki67-positive cells was carried out (n=6 per group, Ctrl vs sgAnapc10). Statistical analysis was performed using Student's t-test.

Fig 2 Proliferation of tumor cells with ANAPC10 knockdown and HCC formation in Anapc10 knockout mice

图3 ANAPC10 表达相关的通路富集分析及稳转细胞系和小鼠肝癌组织的WB和IHC验证Note：A. Patients from the TCGA_LIHC and CHCC_HBV cohorts were divided into ANAPC10-high-expression and ANAPC10-low-expression groups according to their mRNA levels. In the TCGA_LIHC cohort, there were 181 patients in the ANAPC10-high group and 190 in the ANAPC10-low group. In the CHCC_HBV cohort, there were 91 patients in the ANAPC10-high group and 68 in the ANAPC10-low group. Differentially expressed genes (DEGs) between the high- and low-expression groups (ranked by P value) were identified in both datasets, and 2 912 overlapping DEGs were found across the cohorts. B/C. KEGG (B) pathway and Gene Ontology Biological Process (GOBP) (C) enrichment analyses were carried out on the 2 912 common DEGs. D. Gene Set Enrichment Analysis (GSEA, version 4.3.2) was performed on CHCC_HBV patients stratified by ANAPC10 expression (91 patients in the ANAPC10-high group and 68 in the ANAPC10-low group). E/F. Protein expression levels of p-AKT and p-S6K in ANAPC10-knockdown SNU-398 (E) and MHCC-97H (F) cell lines were determined. G. Protein expression levels of p-Akt and p-S6k in the tumor tissues of the Anapc10-knockout orthotopic mouse hepatocellular carcinoma model were measured. H. Immunohistochemical (IHC) staining of p-S6k protein was conducted in the tumor tissues of the orthotopic hepatocellular carcinoma mouse model, along with relative IHC scoring statistics (n=6). Scale bar=100 µm. Statistical analysis was performed using the t-test.

Fig 3 Pathway enrichment analysis associated with ANAPC10 expression and WB/IHC validation in stable cell lines and mouse liver cancer tissues

图4 稳转细胞系和小鼠肝癌组织中脂代谢关键蛋白的WB和IHC验证Note： A/B. Validation of changes in the protein expression of FASN and SCD1 in ANAPC10-knockdown stable cell lines (SNU-398 and MHCC-97H). C. Verification of the protein levels of Fasn and Scd1 in Anapc10-knockout orthotopic mouse HCC tumors. D. IHC staining of Fasn and Scd1 proteins in orthotopic HCC tumor tissues from mouse models, along with the corresponding quantitative IHC scores (n=6 biological replicates per group). E. Oil Red O staining of lipid droplets in the orthotopic HCC tumor tissues, accompanied by quantitative analysis of lipid accumulation (n=6 biological replicates per group). Scale bar=100 μm. Statistical analysis was carried out using the t-test.

Fig 4 WB and IHC validation of key lipid metabolism proteins in stable cell lines and murine hepatocellular carcinoma tissues