Journal: PLoS ONE

Article Title: Soluble factors from adipose tissue-derived mesenchymal stem cells promote canine hepatocellular carcinoma cell proliferation and invasion

doi: 10.1371/journal.pone.0191539

Figure Lengend Snippet: (A, B) Representative images of Transwell assays observed under a light microscope in the cultures without (A) and (B) with AT-MSC-CM. (C) The percent of invading HCC cells was significantly increased in cells cultured with AT-MSC-CM compared with control cells cultured without AT-MSC-CM. Data are expressed as mean ± SD. ** P < 0.01.

Article Snippet: AZACH (Cosmo Bio, Tokyo, Japan), a canine HCC cell line, was cultured in H-DMEM supplemented with 10% FBS and 1% antibiotic–antimycotic solution in a humidified atmosphere of 5% CO 2 at 37°C.

Techniques: Light Microscopy, Cell Culture, Control

Journal: PLoS ONE

Article Title: Soluble factors from adipose tissue-derived mesenchymal stem cells promote canine hepatocellular carcinoma cell proliferation and invasion

doi: 10.1371/journal.pone.0191539

Figure Lengend Snippet: Expression of mRNA of the indicated growth factors and MMPs were evaluated in canine HCC cells cultured with AT-MSC-CM compared with control HCC cells cultured without AT-MSC-CM. Data are expressed as mean ± SD. * P < 0.05; ** P < 0.01.

Article Snippet: AZACH (Cosmo Bio, Tokyo, Japan), a canine HCC cell line, was cultured in H-DMEM supplemented with 10% FBS and 1% antibiotic–antimycotic solution in a humidified atmosphere of 5% CO 2 at 37°C.

Techniques: Expressing, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: eIF4A1 Is a Prognostic Marker and Actionable Target in Human Hepatocellular Carcinoma

doi: 10.3390/ijms24032055

Figure Lengend Snippet: Effects of the eIF4A inhibitors, CR-1-31-B and Zotatifin, in HCC cell lines on the levels of elements of oncogenic pathways involved in hepatocarcinogenesis. Western blot analysis was used to assess the levels of several effectors of oncogenic cascades in HLE ( A ) and HLF ( B ) cell lines exposed to increasing concentrations of CR-1-31-B and Zotatifin inhibitors (0, 10, 20, 50, and 100 nM). Cells were treated for 48 h, and western blot analysis was conducted at this time point.

Article Snippet: The human HCC cell lines HLE (JCRB0404, Xenotech, Kansas City, MO, USA), HLF (JCRB0405, Tebu-bio GmbH, Offenbach, Germany), PLC/PRF/5 (300315, CLS Cell lines Service GmbH, Eppelheim, Germany), SNU182 (CRL-2235, LGC Standards GmbH), and SNU449 (CRL-2234, LGC Standards GmbH) were cultured at 37 °C in a 5% CO 2 humidified atmosphere.

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: eIF4A1 Is a Prognostic Marker and Actionable Target in Human Hepatocellular Carcinoma

doi: 10.3390/ijms24032055

Figure Lengend Snippet: Complete inactivation of mTOR effectors after synergistic eIF4A and mTOR inhibition using CR-1-31-B and Zotatifin in combination with Rapalink-1 in HCC cell lines. Western blot analysis in HLE and HLF cell lines challenged with CR-1-31-B (10 nM) and Zotatifin (10 nM) and Rapalink-1 (10 nM) inhibitors, alone or in combination (1:1). Cells were treated for 48 h, and western blot analysis was conducted at this time point.

Article Snippet: The human HCC cell lines HLE (JCRB0404, Xenotech, Kansas City, MO, USA), HLF (JCRB0405, Tebu-bio GmbH, Offenbach, Germany), PLC/PRF/5 (300315, CLS Cell lines Service GmbH, Eppelheim, Germany), SNU182 (CRL-2235, LGC Standards GmbH), and SNU449 (CRL-2234, LGC Standards GmbH) were cultured at 37 °C in a 5% CO 2 humidified atmosphere.

Techniques: Inhibition, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Dysadherin/YAP axis fuels stem plasticity and immune escape in liver cancer

doi: 10.1038/s41392-025-02520-4

Figure Lengend Snippet: Dysadherin promotes cancer stem-like cell features and tumor progression in HCC. a UMAP plot of single-cell transcriptomes from HCC patient samples ( GSE166635 ; n = 2). Cell lineage trajectories were inferred using Monocle2, highlighting the progression toward a malignant state. The expression of CSC- and malignancy-associated genes was mapped along the trajectory. b UMAP and GSEA of tumor clusters from GSE166635 , comparing dysadherin high ( n = 1647) and dysadherin low ( n = 2853) tumor cells. c GSEA of DEGs between dysadherin high and dysadherin low tumors from bulk RNA-seq data ( GSE9843 ; FDR < 0.05). d Violin plots showing FXYD5 expression levels in normal liver tissue ( n = 50) and primary HCC tissues ( n = 371; TCGA-LIHC), and stratified by tumor grades. e Kaplan–Meier survival analysis for overall survival based on FXYD5 expression in TCGA-LIHC cohort. Statistical significance was assessed by log-rank tests. f Limiting dilution sphere formation assay (left) and Aldefluor assay (right) evaluating the effect of dysadherin expression on CSC properties. Scale bar = 50 μm. g Heatmaps of CSC- and malignancy-related gene expression in HCC cells with dysadherin overexpression or knockdown. h In vivo limiting dilution assay (LDA) assessing the tumor-initiating potential of SK-Hep1 cells with or without dysadherin-knockdown (DYS KD ). i Tumor growth curves from the in vivo limiting dilution assay comparing tumor-initiating capacity of DYS KD versus control (WT) SK-Hep1 cells across multiple inoculation doses (0.5–10 × 10 3 cells/mouse). j Heatmaps showing the expression of CSC-associated genes in dysadherin KD versus control SK-Hep1 cells. k Schematic view of DEN/CCl 4 -induced HCC mouse model. l Representative gross liver images and immunofluorescence staining of liver sections from Fxyd5 +/+ and Fxyd5 −/− mice. Scale bar = 200 μm. m Expression of HCC markers in tumor (T) and paired adjacent normal tissues (P) from DEN/CCl 4 -treated mice. n Quantification of tumor number and distribution of tumor sizes, including largest tumor per mouse. o Tumor incidence in mice at the indicated time points. p Heatmap showing expression levels of CSC- and malignancy-associated genes in liver tissues from mice. Data are presented as means ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t-tests and one-way ANOVA with Dunnett’s multiple comparison tests. *, ** and *** indicate p < 0.05, p < 0.01, and p < 0.001

Article Snippet: The human HCC cell lines, HepG2, SNU368, and SK-Hep1 were obtained from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea).

Techniques: Expressing, RNA Sequencing, Tube Formation Assay, Gene Expression, Over Expression, Knockdown, In Vivo, Limiting Dilution Assay, Control, Immunofluorescence, Staining, Two Tailed Test, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: Dysadherin/YAP axis fuels stem plasticity and immune escape in liver cancer

doi: 10.1038/s41392-025-02520-4

Figure Lengend Snippet: Dysadherin acts as an upstream regulator of YAP signaling to promote malignancy in HCC. a GSEA of DEGs between dysadherin high and dysadherin low HCC tumors ( GSE9843 ), highlighting enrichment of YAP-related oncogenic signatures. b Left: UMAP plot of tumor cell clusters from GSE166635 , color-coded by FXYD5 expression. Right: UMAPs and violin plots showing YAP signature scores and expression of YAP target genes ( CTGF , CYR61 ) in FXYD5 high versus FXYD5 low clusters. c Kaplan–Meier survival analysis of overall survival in TCGA-LIHC cohort stratified by high or low expression of FXYD5 and YAP target genes ( CTGF or CYR61 ). Significance was assessed by log-rank test. d Immunofluorescence (IF) staining of dysadherin and nuclear YAP in paired normal and tumor tissues from HCC patients ( n = 14). Representative images and quantification of nuclear YAP signal intensity are shown. Scale bar = 100 μm. e Violin plots comparing dysadherin expression and nuclear YAP intensity across histological grades of HCC in patient tissues ( n = 14). f RT-qPCR and immunoblot analyses of dysadherin, total YAP, and active YAP expression in normal hepatic (THLE-3) and HCC cell lines. g Heatmaps and immunofluorescence staining showing expression of YAP target genes and subcellular localization of active YAP in dysadherin-overexpressing (OE) and knockdown (KD) cells. Scale bar = 10 μm. h Immunoblotting analysis of active YAP (non-phosphorylated) and phospho-YAP (S127) following dysadherin OE or KD. i Heatmap showing YAP target gene expression in tumor from DEN/CCl₄-treated Fxyd5 +/+ and Fxyd5 −/− mice. j Immunoblotting of YAP pathway components and downstream targets (CTGF) in liver lysates from Fxyd5 +/+ and Fxyd5 −/− mice. Data are presented as means ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t-tests and one-way ANOVA with Dunnett’s multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The human HCC cell lines, HepG2, SNU368, and SK-Hep1 were obtained from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea).

Techniques: Expressing, Immunofluorescence, Staining, Quantitative RT-PCR, Western Blot, Knockdown, Targeted Gene Expression, Two Tailed Test, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: Dysadherin/YAP axis fuels stem plasticity and immune escape in liver cancer

doi: 10.1038/s41392-025-02520-4

Figure Lengend Snippet: Dysadherin promotes CSC features through YAP activation in HCC. a Upstream regulator analysis using dysadherin-correlated transcriptional signatures in HCC tumors ( GSE9843 ), predicting YAP as a central effector. b Immunofluorescence (IF) staining showing active YAP localization in spheroid cultures of dysadherin-overexpressing (OE) or knockdown (KD) HCC cells (PLC/PRF/5 and SK-Hep1). Scale bar = 100 μm. c Heatmaps showing the expression of YAP target genes in tumors derived from the LDA experiment. d Immunoblot analysis of active and total YAP, phospho-YAP (S127), and CTGF expression in tumors derived from the LDA experiment. e Immunoblot analysis showing YAP activation status in dysadherin OE or KD cells treated with YAP knockdown (shYAP), verteporfin (VP), or constitutively active YAP (YAP5SA). f , i , l Sphere formation assays showing size and growth of spheroids under the indicated conditions (shYAP, verteporfin, or YAP5SA). Scale bar = 100 μm. g , j , m Clonogenic survival assays measuring colony-forming ability in PLC/PRF/5 or SK-Hep1 cells. h , k , n Heatmaps showing expression of CSC-associated genes in dysadherin OE or KD cells. Data are presented as means ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t-tests and one-way ANOVA with Dunnett’s multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The human HCC cell lines, HepG2, SNU368, and SK-Hep1 were obtained from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea).

Techniques: Activation Assay, Immunofluorescence, Staining, Knockdown, Expressing, Derivative Assay, Western Blot, Two Tailed Test, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: Dysadherin/YAP axis fuels stem plasticity and immune escape in liver cancer

doi: 10.1038/s41392-025-02520-4

Figure Lengend Snippet: The dysadherin–FAK–YAP axis drives TEAD2-dependent transcription of pluripotency genes in HCC. a GSEA of DEGs from dysadherin high versus dysadherin low HCC tumors ( GSE9843 ). b , c Immunoblot analyses showing levels of phosphorylated FAK (p-FAK), total and active YAP, phospho-YAP (S127), and pluripotency transcription factors (OCT4, KLF4, MYC, SOX2) in PLC/PRF/5 and SK-Hep1 cells. Cells were subjected to dysadherin OE or KD and treated with shYAP, constitutively active YAP mutant (YAP5SA), or the FAK inhibitor PND-1186. d Immunoblot analysis showing levels of dysadherin (DYS), phosphorylated FAK (P-FAK), total FAK (T-FAK), phosphorylated LATS1/2 (P-LATS1/2), total LATS1, phosphorylated MST1/2 (P-MST1/2), and total MST1 in PLC/PRF/5 cells. Cells were subjected to dysadherin overexpression with or without a FAK inhibitor. e RT-qPCR analysis of pluripotency genes in SK-Hep1 cells expressing YAP5SA or treated with verteporfin. f Luciferase reporter assay assessing OCT4 promoter activity in dysadherin OE PLC/PRF/5 cells treated with or without verteporfin. g Venn diagram illustrating overlap of TEAD2 and TEAD4 as candidate transcriptional regulators of OCT4 from the EPD and GTRD databases. h RT-qPCR analysis of OCT4 mRNA levels in YAP5SA-expressing SK-Hep1 cells following siRNA-mediated knockdown of TEAD2 or TEAD4. i ChIP-qPCR confirming direct binding of TEAD2 to the OCT4 promoter region using seven primer sets spanning from −1400 to +9 bp relative to the transcription start site. For ChIP-Re-ChIP analysis in SK-Hep1 cells, chromatin was first immunoprecipitated with an anti-TEAD2 antibody, and the resulting material was re-immunoprecipitated with an anti-YAP antibody. PCR was performed with primers spanning the OCT4 promoter. IgG was used as a negative control. Data are presented as means ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t-tests or one-way ANOVA with Dunnett’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The human HCC cell lines, HepG2, SNU368, and SK-Hep1 were obtained from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea).

Techniques: Western Blot, Mutagenesis, Over Expression, Quantitative RT-PCR, Expressing, Luciferase, Reporter Assay, Activity Assay, Knockdown, ChIP-qPCR, Binding Assay, Immunoprecipitation, Negative Control, Two Tailed Test, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: Dysadherin/YAP axis fuels stem plasticity and immune escape in liver cancer

doi: 10.1038/s41392-025-02520-4

Figure Lengend Snippet: The dysadherin–YAP axis modulates drug resistance and PD-L1–mediated immune evasion in HCC. a GSEA of DEGs between dysadherin high and dysadherin low tumors ( GSE9843 ), revealing enrichment of immunotherapy resistance and drug resistance signatures. b Heatmaps showing increased expression of gene sets associated with resistance to tyrosine-kinase inhibitors (left), cytostatic drugs (middle), and doxorubicin (right) in dysadherin high HCC tumors. c Cell viability and IC 50 curves of PLC/PRF/5 (dysadherin OE) and SK-Hep1 (dysadherin KD) cells treated with verteporfin or YAP5SA, in the presence of sorafenib. d Apoptosis analysis using Annexin V/PI staining in PLC/PRF/5 and SK-Hep1 cells under the same conditions as in ( c ). e Heatmap showing elevated expression of immunosuppressive genes and immune checkpoint–related transcripts in dysadherin high tumors from GSE9843 . f Immunoblot analysis of active YAP, phospho-YAP (S127), total YAP, and PD-L1 in dysadherin-modified cells treated with shYAP, verteporfin, or YAP5SA. g ChIP-qPCR showing TEAD2 binding to the PD-L1 promoter region in dysadherin-OE PLC/PRF/5 cells. ChIP-Re-ChIP analysis showing co-occupancy of TEAD2 and YAP on the PD-L1 promoter in SK-Hep1 cells. Sequential immunoprecipitation was performed first with an anti-TEAD2 antibody, followed by an anti-YAP antibody. IgG served as a negative control. h Immunofluorescence analysis of PD-L1 and PD-1 binding in dysadherin-OE and KD cells using PD-1-Fc fusion protein staining. Scale bar = 100 μm. Flow cytometry analysis of CD69 expression ( i ) and the measurement of IFN-γ secretion ( j ) in Jurkat T cells co-cultured with dysadherin-modified HCC cells, treated with verteporfin or YAP5SA under CD3/CD28 stimulation. Data are presented as means ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t-tests and one-way ANOVA with Dunnett’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The human HCC cell lines, HepG2, SNU368, and SK-Hep1 were obtained from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea).

Techniques: Expressing, Staining, Western Blot, Modification, ChIP-qPCR, Binding Assay, Immunoprecipitation, Negative Control, Immunofluorescence, Flow Cytometry, Cell Culture, Two Tailed Test, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: Dysadherin/YAP axis fuels stem plasticity and immune escape in liver cancer

doi: 10.1038/s41392-025-02520-4

Figure Lengend Snippet: The dysadherin–YAP axis drives HCC progression and facilitates immune evasion in vivo. a Schematic overview of the humanized mouse model (Hu-NSG-SGM3) used for in vivo tumor studies. b Tumor growth of SK-Hep1 cells with or without dysadherin knockdown (DYS KD ) in the humanized mouse model. Immunofluorescence ( c , e ) and immunoblot ( d ) analysis of dysadherin, YAP, CTGF, and PD-L1 expression in SK-Hep1 tumors. Scale bar = 100 μm. f Left: Flow cytometric quantification of tumor-infiltrating CD8⁺ T cells and PD1⁺TIM3⁺ exhausted T cells. Right: Heatmap of cytotoxic and exhaustion marker gene expression profiles of T cells in DYS KD versus control tumors. g Schematic overview of the in vivo peptide treatment protocol in NSG mice. Representative tumor images ( h ) and quantification of tumor growth and weight ( i ) in SK-Hep1 xenografts treated with the dysadherin-inhibitory peptide versus vehicle (DMSO) for 28 days (n = 6 mice/group). j Immunoblotting of dysadherin, YAP activation markers, and CTGF expression in tumor tissues after peptide treatment. k Experimental design for splenic injection model of liver metastasis using PLC/PRF/5 spheroids with or without dysadherin OE and shYAP. Scale bar = 1 mm. Quantification of tumor incidence ( l ), tumor size ( m ) in the liver metastasis model. n Schematic diagram of integrin-FAK-YAP-TEAD axis-mediated stemness acquisition and immune escape in liver cancer. Created with BioRender.com. Data are presented as means ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t-tests and one-way ANOVA with Dunnett’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The human HCC cell lines, HepG2, SNU368, and SK-Hep1 were obtained from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea).

Techniques: In Vivo, Knockdown, Immunofluorescence, Western Blot, Expressing, Marker, Gene Expression, Control, Activation Assay, Injection, Two Tailed Test, Comparison