Journal: International Journal of Oncology

Article Title: Inhibiting SSBP1 enhances ferroptosis and improves the effectiveness of sorafenib treatment for liver cancer

doi: 10.3892/ijo.2025.5778

Figure Lengend Snippet: Integrated analysis of the expression of SSBP1 in liver cancer. (A) Distribution of differential gene expression levels after mapping mitochondrial-located genes (MitoCarta3.0 list) onto the TCGA-LIHC dataset. (B) Enrichment analysis using the R language. The gene sets ranking at the top were predominantly implicated in mitochondrial organization and biogenesis. (C) mRNA levels of mitochondrial biogenesis genes (including ALAS1, SIRT3, TFAM, TFB2M, SSBP1, CYCS, POLRMT and POLG) in liver cancer and paracancerous tissue (n=5). (D) Expression profiles of POLG and SSBP1 in patients with liver cancer and corresponding controls derived from the GSE14520 (tumor, n=225; non-tumor, n=220) and GSE101685 datasets. (tumor, n=24; non-tumor, n=8). (E) Transcriptional expression levels of SSBP1 in the TCGA-LIHC dataset. (F) Association between SSBP1 expression and the prognosis of liver cancer in the overall population (n=364) using the Kaplan Meier Plotter online survival analysis tool. SSBP1, single-stranded DNA binding protein 1; TCGA-LIHC, The Cancer Genome Atlas liver hepatocellular carcinoma; ALAS1, aminolevulinic acid synthase 1; SIRT3, sirtuin 3; TFAM, transcription factor A, mitochondrial; TFB2M, transcription factor B2, mitochondrial; CYCS, cytochrome c, somatic; POLRMT, RNA polymerase mitochondrial; POLG, DNA polymerase γ; TPM, transcripts per million.

Article Snippet: Following blocking with 10% skimmed milk powder solution for 2 h at room temperature, the PVDF membrane was incubated with primary antibodies against SSBP1 (anti-rabbit; cat. no. 12212-1-AP), voltage-dependent anion channel (VDAC; anti-rabbit; cat. no. 10866-1-AP; both 1:1,000; both Proteintech Group, Inc.), Flag (anti-mouse; cat. no. F1804; Merck KGaA; 1:10,000), translocase of the outer mitochondrial membrane 20 (TOM20; anti-rabbit; cat. no. A19403; 1:5,000), glutathione peroxidase 4 (GPX4; anti-rabbit; cat. no. A11243), SLC7A11 (anti-rabbit; cat. no. A13685; both 1:1,000), and β-actin (anti-rabbit; cat. no. AC026; 1:100,000; all from ABclonal Biotech Co., Ltd.) at 4°C overnight.

Techniques: Expressing, Gene Expression, Derivative Assay, Binding Assay

Journal: International Journal of Oncology

Article Title: Inhibiting SSBP1 enhances ferroptosis and improves the effectiveness of sorafenib treatment for liver cancer

doi: 10.3892/ijo.2025.5778

Figure Lengend Snippet: Knockout of SSBP1 in Hep3B cells inhibits the proliferation and migratory capacity of liver cancer cells. (A) SSBP1 expression in control and SSBP1-KO Hep3B cells. (B) MTT assay showed the relative viability of control and SSBP1-KO Hep3B cells (n=10). (C) Representative colony formation plots showing the proliferation capacity of Hep3B cells with SSBP1 KO. (D) Colony formation capacity (n=4). (E) Migratory capacity of control and SSBP1-KO Hep3B cells was examined by Transwell assay. (F) Wound healing assay was used to determine the migratory capacity of Hep3B cells with SSBP1 deficiency. Scale bar, 200 μ m. Magnification, ×40. SSBP1, single-stranded DNA binding protein 1; SSBP1-KO, (single-stranded DNA binding protein 1) knockout.

Article Snippet: Following blocking with 10% skimmed milk powder solution for 2 h at room temperature, the PVDF membrane was incubated with primary antibodies against SSBP1 (anti-rabbit; cat. no. 12212-1-AP), voltage-dependent anion channel (VDAC; anti-rabbit; cat. no. 10866-1-AP; both 1:1,000; both Proteintech Group, Inc.), Flag (anti-mouse; cat. no. F1804; Merck KGaA; 1:10,000), translocase of the outer mitochondrial membrane 20 (TOM20; anti-rabbit; cat. no. A19403; 1:5,000), glutathione peroxidase 4 (GPX4; anti-rabbit; cat. no. A11243), SLC7A11 (anti-rabbit; cat. no. A13685; both 1:1,000), and β-actin (anti-rabbit; cat. no. AC026; 1:100,000; all from ABclonal Biotech Co., Ltd.) at 4°C overnight.

Techniques: Knock-Out, Expressing, Control, MTT Assay, Transwell Assay, Wound Healing Assay, Binding Assay

Journal: International Journal of Oncology

Article Title: Inhibiting SSBP1 enhances ferroptosis and improves the effectiveness of sorafenib treatment for liver cancer

doi: 10.3892/ijo.2025.5778

Figure Lengend Snippet: SSBP1-OE enhances proliferation and migratory capacity of liver cancer cells. (A) Representative bands of control and SSBP1-OE Hep3B cells to validate the effect by Western blotting. Flag signals represent fusion proteins detected by anti-Flag antibody. Control vector-transfected cells were used as negative controls to confirm specific expression of the fusion protein. (B) Viability of control and SSBP1-OE Hep3B cells (n=10). (C) Representative colony formation plots showing the proliferation capacity of Hep3B cells with SSBP1 OE. (D) Colony formation capacity (n=4). (E) Migratory capacity of control and SSBP1-OE Hep3B cells was examined by Transwell assay. (F) Wound healing assay was used to determine the migratory capacity of Hep3B cells with SSBP1 OE. Scale bar, 200 μ m. Magnification, ×40. SSBP1-OE, (single-stranded DNA binding protein 1) overexpression.

Article Snippet: Following blocking with 10% skimmed milk powder solution for 2 h at room temperature, the PVDF membrane was incubated with primary antibodies against SSBP1 (anti-rabbit; cat. no. 12212-1-AP), voltage-dependent anion channel (VDAC; anti-rabbit; cat. no. 10866-1-AP; both 1:1,000; both Proteintech Group, Inc.), Flag (anti-mouse; cat. no. F1804; Merck KGaA; 1:10,000), translocase of the outer mitochondrial membrane 20 (TOM20; anti-rabbit; cat. no. A19403; 1:5,000), glutathione peroxidase 4 (GPX4; anti-rabbit; cat. no. A11243), SLC7A11 (anti-rabbit; cat. no. A13685; both 1:1,000), and β-actin (anti-rabbit; cat. no. AC026; 1:100,000; all from ABclonal Biotech Co., Ltd.) at 4°C overnight.

Techniques: Control, Western Blot, Plasmid Preparation, Transfection, Expressing, Transwell Assay, Wound Healing Assay, Binding Assay, Over Expression

Journal: International Journal of Oncology

Article Title: Inhibiting SSBP1 enhances ferroptosis and improves the effectiveness of sorafenib treatment for liver cancer

doi: 10.3892/ijo.2025.5778

Figure Lengend Snippet: SSBP1 deficiency inhibits mitochondrial function. (A) qPCR was used to analyze the mtDNA copy number of liver cancer and paracancerous tissue (n=6). (B) Immunofluorescence was performed to detect the mitochondrial morphology in Hep3B cells following SSBP1 KO. Scale bar, 10 μ m. Magnification, ×630. (C) Flow cytometry was employed to analyze the mitochondrial mass of control and SSBP1-KO Hep3B cells (n=6-7). (D) qPCR was used to analyze the mtDNA copy number of control and SSBP1-KO Hep3B cells (n=7). (E) Expression of mitochondrial proteins in control and SSBP1-KO Hep3B cells. (F) Flow cytometry was employed to analyze (G) levels of ROS of control and SSBP1-KO Hep3B cells (n=9). (H) Flow cytometry was applied to analyze (I) MMP of control and SSBP1-KO Hep3B cells (n=3). q, Quantitative; KO, SSBP1 (single-stranded DNA binding protein 1) knockout; mtDNA, mitochondrial DNA; VDAC, Voltage-Dependent Anion Channel; TOM20, Translocase of the Outer Mitochondrial Membrane 20; ROS, Reactive Oxygen Species.

Article Snippet: Following blocking with 10% skimmed milk powder solution for 2 h at room temperature, the PVDF membrane was incubated with primary antibodies against SSBP1 (anti-rabbit; cat. no. 12212-1-AP), voltage-dependent anion channel (VDAC; anti-rabbit; cat. no. 10866-1-AP; both 1:1,000; both Proteintech Group, Inc.), Flag (anti-mouse; cat. no. F1804; Merck KGaA; 1:10,000), translocase of the outer mitochondrial membrane 20 (TOM20; anti-rabbit; cat. no. A19403; 1:5,000), glutathione peroxidase 4 (GPX4; anti-rabbit; cat. no. A11243), SLC7A11 (anti-rabbit; cat. no. A13685; both 1:1,000), and β-actin (anti-rabbit; cat. no. AC026; 1:100,000; all from ABclonal Biotech Co., Ltd.) at 4°C overnight.

Techniques: Immunofluorescence, Flow Cytometry, Control, Expressing, Binding Assay, Knock-Out, Membrane

Journal: International Journal of Oncology

Article Title: Inhibiting SSBP1 enhances ferroptosis and improves the effectiveness of sorafenib treatment for liver cancer

doi: 10.3892/ijo.2025.5778

Figure Lengend Snippet: SSBP1 deficiency triggers ferroptosis in liver cancer cells. (A) Flow cytometry was performed to analyze (B) relative death level (PI + ) in control and SSBP1 -KO Hep3B cells (n=8-10). (C) Flow cytometry was applied to analyze (D) C11-BODIPY levels of control and SSBP1 -KO Hep3B cells (n=8). (E) Fluorescence images of C11-BODIPY-stained Hep3B cells. (F) Relative fluorescence intensity (n=3). Scale bar, 20 μ m. Magnification, ×200. (G) Western blotting of ferroptosis-associated proteins in Control and SSBP1-KO Hep3B cells. (H) mRNA expression of ferroptosis-associated genes in Control and SSBP1-KO Hep3B cells (n=6). SSBP1-KO, SSBP1 (single-stranded DNA binding protein 1) knockout; C11-BODIPY, 11-carbon-substituted boron dipyrromethene; GPX4, glutathione peroxidase 4; SLC7A11, solute carrier family 7 member 11; ME1, malic enzyme 1.

Article Snippet: Following blocking with 10% skimmed milk powder solution for 2 h at room temperature, the PVDF membrane was incubated with primary antibodies against SSBP1 (anti-rabbit; cat. no. 12212-1-AP), voltage-dependent anion channel (VDAC; anti-rabbit; cat. no. 10866-1-AP; both 1:1,000; both Proteintech Group, Inc.), Flag (anti-mouse; cat. no. F1804; Merck KGaA; 1:10,000), translocase of the outer mitochondrial membrane 20 (TOM20; anti-rabbit; cat. no. A19403; 1:5,000), glutathione peroxidase 4 (GPX4; anti-rabbit; cat. no. A11243), SLC7A11 (anti-rabbit; cat. no. A13685; both 1:1,000), and β-actin (anti-rabbit; cat. no. AC026; 1:100,000; all from ABclonal Biotech Co., Ltd.) at 4°C overnight.

Techniques: Flow Cytometry, Control, Fluorescence, Staining, Western Blot, Expressing, Binding Assay, Knock-Out

Journal: International Journal of Oncology

Article Title: Inhibiting SSBP1 enhances ferroptosis and improves the effectiveness of sorafenib treatment for liver cancer

doi: 10.3892/ijo.2025.5778

Figure Lengend Snippet: SSBP1 deficiency enhances the effect of sora treatment. (A) Expression of SSBP1, TOM20 and GPX4 in Con and SSBP1-KO Hep3B cells with or without sora treatment. (B) MTT assay showed the relative viability of Con and SSBP1-KO Hep3B cells with or without sora treatment (n=6). (C) Representative colony formation assay showing proliferation capacity of Hep3B cells with SSBP1 KO in the presence or absence of sora treatment. (D) Colony formation capacity (n=3). (E) Immunofluorescence was performed to detect the mitochondrial morphology in Hep3B cells following SSBP1 KO in the presence or absence of sora. Scale bar, 5 μ m. Magnification, ×630. (F) qPCR was used to analyze the mtDNA copy number of Con and SSBP1-KO Hep3B cells with or without sora treatment (n=4-5). Flow cytometry was employed to analyze (G) mitochondrial mass (n=3-4) and (H) ROS levels (n=4-5) of Con and SSBP1-KO Hep3B cells with or without sora treatment. (I) ROS levels in Con and SSBP1-KO Hep3B cells with or without sora (n=4-5). (J) Flow cytometry was performed to analyze (K) MMP of Control and SSBP1-KO Hep3B cells with or without sora (n=3-4). (L) Fluorescence images of C11-BODIPY-stained Hep3B cells. (M) Relative fluorescence intensity was quantified by ImageJ software (n=3). Scale bar, 20 μ m. Magnification, ×200. (N) mRNA expression of ferroptosis-associated gene GPX4 in Con and SSBP1-KO Hep3B cells in the presence or absence of sora (n=6). TOM20, translocase of the outer mitochondrial membrane 20; GPX4, glutathione peroxidase 4; SSBP1-KO, SSBP1 (single-stranded DNA binding protein 1) knockout; mtDNA, mitochondrial DNA; Con, control; sora, sorafenib.

Article Snippet: Following blocking with 10% skimmed milk powder solution for 2 h at room temperature, the PVDF membrane was incubated with primary antibodies against SSBP1 (anti-rabbit; cat. no. 12212-1-AP), voltage-dependent anion channel (VDAC; anti-rabbit; cat. no. 10866-1-AP; both 1:1,000; both Proteintech Group, Inc.), Flag (anti-mouse; cat. no. F1804; Merck KGaA; 1:10,000), translocase of the outer mitochondrial membrane 20 (TOM20; anti-rabbit; cat. no. A19403; 1:5,000), glutathione peroxidase 4 (GPX4; anti-rabbit; cat. no. A11243), SLC7A11 (anti-rabbit; cat. no. A13685; both 1:1,000), and β-actin (anti-rabbit; cat. no. AC026; 1:100,000; all from ABclonal Biotech Co., Ltd.) at 4°C overnight.

Techniques: Expressing, MTT Assay, Colony Assay, Immunofluorescence, Flow Cytometry, Control, Fluorescence, Staining, Software, Membrane, Binding Assay, Knock-Out

Journal: International Journal of Oncology

Article Title: Inhibiting SSBP1 enhances ferroptosis and improves the effectiveness of sorafenib treatment for liver cancer

doi: 10.3892/ijo.2025.5778

Figure Lengend Snippet: SSBP1 deficiency enhances liver cancer cell sensitivity to sorafenib. SSBP1 deficiency impairs mitochondrial function and exacerbates lipid peroxidation in liver cancer cells, sensitizing them to sorafenib-induced ferroptosis. SSBP1-KO-driven mitochondrial dysfunction and cytosolic/mitochondrial lipid peroxide accumulation leads to diminished GPX4 and SLC7A11 activity. These alterations amplify ferroptosis under sorafenib treatment, underscoring SSBP1 loss as a key determinant of sorafenib sensitivity in liver cancer by disrupting redox homeostasis and mitochondrial integrity. MMP, mitochondrial membrane potential; SSBP1-KO, single-stranded DNA binding protein 1-knockout; GPX4, glutathione peroxidase 4; SLC7A11, solute carrier family 7 member 11; WT, wildtype.

Article Snippet: Following blocking with 10% skimmed milk powder solution for 2 h at room temperature, the PVDF membrane was incubated with primary antibodies against SSBP1 (anti-rabbit; cat. no. 12212-1-AP), voltage-dependent anion channel (VDAC; anti-rabbit; cat. no. 10866-1-AP; both 1:1,000; both Proteintech Group, Inc.), Flag (anti-mouse; cat. no. F1804; Merck KGaA; 1:10,000), translocase of the outer mitochondrial membrane 20 (TOM20; anti-rabbit; cat. no. A19403; 1:5,000), glutathione peroxidase 4 (GPX4; anti-rabbit; cat. no. A11243), SLC7A11 (anti-rabbit; cat. no. A13685; both 1:1,000), and β-actin (anti-rabbit; cat. no. AC026; 1:100,000; all from ABclonal Biotech Co., Ltd.) at 4°C overnight.

Techniques: Activity Assay, Membrane, Binding Assay, Knock-Out