Journal: BMC Cancer

Article Title: Unveiling the ITGβ1-CLIC1 axis: a new frontier in oral squamous cell carcinoma progression and immune evasion: insights from the tumor-mimetic ECM system

doi: 10.1186/s12885-025-15402-3

Figure Lengend Snippet: Immune-related analysis of the CLIC1 and ITGB1. A Correlation heatmap of eleven genes with 22 immune cell types. B Heatmap of the proportion of 22 immune cells across samples. C CLIC1 gene expression differences in 22 immune cells between high- and low-expression groups. D ITGB1 gene expression differences in 22 immune cells between high- and low-expression groups. E Partial Spearman correlation between ITGB1 and T cell follicular helper, adjusted for purity via TIMER. F Partial Spearman correlation between CLIC1 and T cell follicular helper, adjusted for purity via TIMER

Article Snippet: Specific primary antibodies were used to detect the target proteins: ITGβ1 (1:1000; Wanleibio, Shenyang, China; Rabbit anti-human) and CLIC1 (1:1000; Wanleibio, Shenyang, China; Rabbit anti-human), followed by incubation with secondary antibody (Goat anti-rabbit).

Techniques: Gene Expression, Expressing

Journal: BMC Cancer

Article Title: Unveiling the ITGβ1-CLIC1 axis: a new frontier in oral squamous cell carcinoma progression and immune evasion: insights from the tumor-mimetic ECM system

doi: 10.1186/s12885-025-15402-3

Figure Lengend Snippet: Experimental validation of IGTβ1 and CLIC1 in normal and OSCC samples. A H&E staining. B-D IHC staining, and positive cell count of IGTβ1 and CLIC1. E-G Protein expression of IGTβ1 and CLIC1 by WB. H Gene expression by qPCR. (Scale bar, 100x. ** p <0.01, *** p <0.001)

Article Snippet: Specific primary antibodies were used to detect the target proteins: ITGβ1 (1:1000; Wanleibio, Shenyang, China; Rabbit anti-human) and CLIC1 (1:1000; Wanleibio, Shenyang, China; Rabbit anti-human), followed by incubation with secondary antibody (Goat anti-rabbit).

Techniques: Biomarker Discovery, Staining, Immunohistochemistry, Cell Counting, Expressing, Gene Expression

Journal: BMC Cancer

Article Title: Unveiling the ITGβ1-CLIC1 axis: a new frontier in oral squamous cell carcinoma progression and immune evasion: insights from the tumor-mimetic ECM system

doi: 10.1186/s12885-025-15402-3

Figure Lengend Snippet: Analysis of clinical samples and data from OSCC. A Patients screening process. B Baseline characteristics of patients. C Correlation between ITGβ1 and CLIC1 expression and stage. D Recurrence-free survival analysis

Article Snippet: Specific primary antibodies were used to detect the target proteins: ITGβ1 (1:1000; Wanleibio, Shenyang, China; Rabbit anti-human) and CLIC1 (1:1000; Wanleibio, Shenyang, China; Rabbit anti-human), followed by incubation with secondary antibody (Goat anti-rabbit).

Techniques: Expressing

Journal: BMC Cancer

Article Title: Unveiling the ITGβ1-CLIC1 axis: a new frontier in oral squamous cell carcinoma progression and immune evasion: insights from the tumor-mimetic ECM system

doi: 10.1186/s12885-025-15402-3

Figure Lengend Snippet: Functions of HSC3 cells treated with CA-dECM. A-D Cell apoptosis. E-F WB analysis of IGTβ1. G-H WB analysis of CLIC1. (** p <0.01)

Article Snippet: Specific primary antibodies were used to detect the target proteins: ITGβ1 (1:1000; Wanleibio, Shenyang, China; Rabbit anti-human) and CLIC1 (1:1000; Wanleibio, Shenyang, China; Rabbit anti-human), followed by incubation with secondary antibody (Goat anti-rabbit).

Techniques:

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: Silencing CLIC1 suppresses glycolysis in gastric cancer cells. ( A ) KEGG pathway enrichment analysis of CLIC1-associated genes. ( B ) Correlation analysis between CLIC1 expression and glycolytic pathway activity. ( C ) qPCR validation of CLIC1 silencing in AGS and HGC27 cells. ( D - E ) Western blot analysis confirmed CLIC1 knockdown efficiency in AGS and HGC27 cells. ( F - G ) ECAR assays showed that CLIC1 silencing decreases glycolytic levels and glycolytic capacity in both cell lines. ( H ) Glucose consumption in CLIC1-silenced AGS and HGC27 cells. ( I ) Intracellular ATP production upon CLIC1 knockdown in both cell lines. ( J ) Lactate secretion in CLIC1-silenced gastric cancer cells. Two-sided unpaired Student’s t-test between two groups, and one-way ANOVA among three or more groups. (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: Expressing, Activity Assay, Biomarker Discovery, Western Blot, Knockdown

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: CLIC1 promoted gastric cancer progression through glycolysis-dependent mechanisms. ( A ) WB confirmed the efficiency of CLIC1 overexpression in AGS and HGC27 gastric cancer cells. ( B – C ) CLIC1 overexpression increased ( B ) glucose consumption and ( C ) lactate secretion, effects that were attenuated by 2-DG. ( D – G ) Wound-healing assays showed that CLIC1 overexpression enhanced cell migration, which was suppressed by 2-DG. ( H – I ) CCK-8 assays demonstrated that CLIC1 overexpression promoted cell proliferation, an effect partially reversed by 2-DG treatment. ( J – M ) Transwell assays revealed that CLIC1 enhanced both migratory and invasive capacities in a glycolysis-dependent manner. ( N ) Representative images of xenograft tumors from each treatment group at the study endpoint. ( O ) Final tumor weights at sacrifice. ( P ) Tumor growth curves showed tumor volume changes over time. One-way ANOVA. (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: Over Expression, Migration, CCK-8 Assay

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: CLIC1 interacts directly with PKM2 in gastric cancer cells. ( A ) Venn diagram showed the overlap among CLIC1-interacting proteins identified by mass spectrometry, glycolysis-related genes, and CLIC1-associated genes from GeneCards and LinkedOmics databases. Eleven overlapping candidates were identified. ( B – C ) Exogenous Co-IP assays in 293T cells demonstrated that CLIC1 immunoprecipitated PKM2 and vice versa, confirming their reciprocal interaction. ( D – E ) Endogenous Co-IP assays in AGS and HGC27 gastric cancer cells further verified the specific interaction between CLIC1 and PKM2. ( F – G ) No interaction was detected between CLIC1 and the PKM1 isoform. ( H ) Immunofluorescence staining showed cytoplasmic co-localization of CLIC1 and PKM2 in AGS and HGC27 cells. ( I ) GST pull-down assay revealed that GST-tagged CLIC1 directly bound to PKM2, whereas the GST control did not. ( J ) Schematic representation of CLIC1 truncation mutants. ( K ) Co-IP assays demonstrated that full-length CLIC1 and two truncation mutants retained PKM2-binding ability

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: Mass Spectrometry, Co-Immunoprecipitation Assay, Immunoprecipitation, Immunofluorescence, Staining, Pull Down Assay, Control, Binding Assay

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: Mapping of the CLIC1–PKM2 interaction domain and analysis of CLIC1’s effect on PKM2 expression and stability. ( A ) Diagram of PKM2 truncation mutants covering different structural domains. ( B ) Co-IP assays showed that deletion of the C-terminal domain abolished the interaction between PKM2 and CLIC1. ( C – F ) Western blot analysis showing that neither CLIC1 overexpression nor knockdown altered PKM2 protein expression levels in AGS and HGC27 gastric cancer cells.( G - H ) CHX chase assay revealed that CLIC1 depletion did not affect PKM2 protein stability. Two-sided unpaired Student’s t-test between two groups, and one-way ANOVA among three or more groups. (* p < 0.05, ** p < 0.01, *** p < 0.001, ns. not significant)

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: Expressing, Co-Immunoprecipitation Assay, Western Blot, Over Expression, Knockdown

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: CLIC1 regulates the oligomeric state and nuclear localization of PKM2. ( A – B ) Protein cross-linking and Western blot analyses showed that CLIC1 overexpression promoted PKM2 dimer formation while reducing tetramer formation in AGS and HGC27 cells. ( C – D ) Silencing of CLIC1 led to decreasing PKM2 dimers and increasing tetramers. ( E – F ) Subcellular fractionation and WB analyses demonstrating that CLIC1 overexpression enhanced the nuclear localization of PKM2. ( G ) Immunofluorescence staining confirmed that CLIC1 overexpression promoted PKM2 accumulation in the nucleus. ( H ) Molecular docking analysis indicated that the binding interface between CLIC1 and PKM2 covered both truncation regions of CLIC1, whereas the interaction site on PKM2 was primarily located within its C-terminal domain

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: Western Blot, Over Expression, Fractionation, Immunofluorescence, Staining, Binding Assay

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: CLIC1 promotes gastric cancer progression by modulating PKM2-mediated glycolysis. ( A ) WB analysis demonstrated PKM2 protein expression following PKM2-IN-1 treatment in AGS and HGC27 cells. ( B – C ) Quantification of lactate production ( B ) and glucose consumption ( C ) in control and CLIC1-overexpressing AGS and HGC27 cells with or without PKM2-IN-1 treatment. ( D ) CCK-8 assay showed that CLIC1 overexpression enhanced gastric cancer cell proliferation, whereas PKM2-IN-1 partially reversed this effect. ( E – H ) Representative images and quantitative analysis of wound-healing assays. ( I – L ) Representative images and statistical quantification of transwell migration and invasion assays. One-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: Expressing, Control, CCK-8 Assay, Over Expression, Migration

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: CLIC1 promotes gastric tumor growth through PKM2-dependent glycolysis in vivo. ( A ) Representative xenograft tumors from each group at the endpoint. ( B ) Tumor weights showed that PKM2-IN-1 treatment attenuated CLIC1-induced tumor growth. ( C ) Tumor growth curves depicted the kinetics of tumor volume expansion over time in each experimental group. ( D ) Representative IHC staining of CLIC1 and PKM2 in tumor tissues. One-way ANOVA. (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: In Vivo, Immunohistochemistry

Journal: Journal of Translational Medicine

Article Title: The CLIC1-PKM2 axis orchestrates glycolytic metabolism to accelerate gastric cancer progression

doi: 10.1186/s12967-025-07463-6

Figure Lengend Snippet: Expression pattern and clinical relevance of PKM2 and CLIC1 in gastric cancer. ( A – B ) Differential expression analysis of CLIC1 ( A ) and PKM2 ( B ) between tumor and adjacent normal tissues in the TCGA-STAD cohort. ( C – D ) Expression levels of CLIC1 ( C ) and PKM2 ( D ) in gastric cancer and normal tissues from the GSE66229 dataset. ( E ) Correlation analysis of CLIC1 and PKM2 expression in the TCGA-STAD cohort. ( F – G ) Kaplan–Meier survival curves showing that high CLIC1 ( F ) and PKM2 ( G ) expression were associated with poor overall survival in gastric cancer patients from the GSE62254 cohort. ( H – J ) Kaplan–Meier plots illustrated that high PKM2 expression correlated with reduced OS, PFS, and PPS. ( K ) RT-qPCR analysis of PKM2 mRNA expression in paired gastric cancer and adjacent normal tissues. ( L ) Western blot analysis of PKM2 protein expression in representative paired GC and normal tissues. ( M – N ) Representative IHC staining and quantification of PKM2 ( M ) and CLIC1 ( N ) in paired gastric cancer specimens. ( O ) Correlation analysis between CLIC1 and PKM2 protein expression levels in gastric cancer tissues. Two-sided unpaired Student’s t-test. (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 (Sigma) for 15 min. After blocking with 5% bovine serum albumin (BSA) for 30 min, cells were incubated overnight at 4 °C with primary antibodies against CLIC1 (#sc-81873, Santa Cruz Biotechnology) and PKM2 (#15822-1-AP, Proteintech).

Techniques: Expressing, Quantitative Proteomics, Quantitative RT-PCR, Western Blot, Immunohistochemistry