Journal: Cell Death Discovery

Article Title: HUWE1 loss promotes stemness and drug resistance in CRC with dysregulated β-catenin destruction complex

doi: 10.1038/s41420-025-02731-2

Figure Lengend Snippet: A The tumor sphere-forming ability of DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion, was examined. Scale bar, 100 µm. B Colony formation assay was performed in DLD1, HT29, and HCT116 cells with or without HUWE1 depletion. C Flow cytometry analysis was performed to assess the ratio of CD24+/CD133+ cells in DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion. D Western blot analysis detected protein levels of LGR5, OCT4, SOX2, and NANOG in DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion. E The IC50 values of oxaliplatin, 5FU, and doxorubicin in DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion, were determined using the CCK-8 assay. F Apoptosis in DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion, was assessed by flow cytometry using Annexin V-FITC and PI dual staining after treatment with 50 µM oxaliplatin, 5FU, or doxorubicin for 48 h. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: DLD1, HCT116, HT29, MCF7, HepG2, HeLa, and A549 cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: Colony Assay, Flow Cytometry, Western Blot, CCK-8 Assay, Staining

Journal: Cell Death Discovery

Article Title: HUWE1 loss promotes stemness and drug resistance in CRC with dysregulated β-catenin destruction complex

doi: 10.1038/s41420-025-02731-2

Figure Lengend Snippet: A The proliferation rate of DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion, was examined using the CCK-8 assay. B EdU proliferation assay was performed in DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion. Scale bar, 100 µm. C Migration capacity of DLD1, HT29, and HCT116, with or without HUWE1 depletion, was examined by wound healing assay. D Transwell invasion assay was performed in DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion. E Immunofluorescence analysis was performed to examine the expression of E-cadherin, N-cadherin, and Vimentin in DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion. Scale bar, 20 µm. F Western blot analysis of DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion, was performed with the indicated antibodies. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: DLD1, HCT116, HT29, MCF7, HepG2, HeLa, and A549 cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: CCK-8 Assay, Proliferation Assay, Migration, Wound Healing Assay, Transwell Invasion Assay, Immunofluorescence, Expressing, Western Blot

Journal: Cell Death Discovery

Article Title: HUWE1 loss promotes stemness and drug resistance in CRC with dysregulated β-catenin destruction complex

doi: 10.1038/s41420-025-02731-2

Figure Lengend Snippet: A Immunofluorescence analysis was performed to detect the expression of β-catenin in the nucleus and cytoplasm of DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion. Scale bar, 20 µm. B Western blot analysis detected the expression of β-catenin, c-Myc, and Cyclin D1 in DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion. C DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion, were transfected with Flag-β-catenin and HA-Ub plasmids. The degree of ubiquitination of β-catenin was analyzed using an immunoprecipitation assay. D DLD1, MCF7, HepG2, HeLa, and A549 cells were transfected with HUWE1 siRNA, and then the expression level of β-catenin was detected by western blot analysis. E DLD1, HT29, and HCT116 cells with HUWE1 depletion were transfected with plasmids encoding for Flag-β-catenin, resHUWE1, full-length APC, and HA-Ub. The degree of ubiquitination of β-catenin was analyzed using an immunoprecipitation assay. F DLD1, HT29, and HCT116 cells with HUWE1 depletion were transfected with plasmids encoding for resHUWE1 and full-length APC, and then expression levels of β-catenin, c-Myc, and Cyclin D1 were detected by western blot analysis. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: DLD1, HCT116, HT29, MCF7, HepG2, HeLa, and A549 cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: Immunofluorescence, Expressing, Western Blot, Transfection, Ubiquitin Proteomics, Immunoprecipitation

Journal: Cell Death Discovery

Article Title: HUWE1 loss promotes stemness and drug resistance in CRC with dysregulated β-catenin destruction complex

doi: 10.1038/s41420-025-02731-2

Figure Lengend Snippet: A Comparison of the interaction between HUWE1 and β-catenin in DLD1 and HT29 cells transfected with control plasmid or full-length APC plasmid using Co-immunoprecipitation assay. B , C Comparison of the interaction between HUWE1 and DVL1 or PPDPF in DLD1, HT29, and HCT116 cells transfected with control or full-length APC plasmids using Co-immunoprecipitation assay. D , E PLA signal was observed in DLD1, HT29, and HCT116 cells transfected with or without full-length APC plasmid by confocal microscopy. Scale bar, 50 µm. F Schematic diagram illustrating a model of HUWE1 protein interactions in the Wnt/β-catenin signaling pathway, depending on the activation status of the β-catenin destruction complex.

Article Snippet: DLD1, HCT116, HT29, MCF7, HepG2, HeLa, and A549 cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: Comparison, Transfection, Control, Plasmid Preparation, Co-Immunoprecipitation Assay, Confocal Microscopy, Activation Assay

Journal: Cell Death Discovery

Article Title: HUWE1 loss promotes stemness and drug resistance in CRC with dysregulated β-catenin destruction complex

doi: 10.1038/s41420-025-02731-2

Figure Lengend Snippet: DLD1 and HCT116 cells, with or without HUWE1 depletion, were transfected with expression plasmids for resHUWE1, full-length APC, or both. A Tumor sphere-forming ability of the indicated cells was examined. Scale bar, 100 µm. B Colony formation assay was performed using the indicated cells. C The proliferation rate of the indicated cells was examined using the CCK-8 assay. D EdU proliferation assay was performed using the indicated cells. Scale bar, 100 µm. E Migration capacity of the indicated cells was examined by wound healing assay. F Transwell invasion assay was performed using the indicated cells. G , H Western blot analysis of the indicated cells was performed using the specified antibodies. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: DLD1, HCT116, HT29, MCF7, HepG2, HeLa, and A549 cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: Transfection, Expressing, Colony Assay, CCK-8 Assay, Proliferation Assay, Migration, Wound Healing Assay, Transwell Invasion Assay, Western Blot

Journal: Cell Death Discovery

Article Title: HUWE1 loss promotes stemness and drug resistance in CRC with dysregulated β-catenin destruction complex

doi: 10.1038/s41420-025-02731-2

Figure Lengend Snippet: A , B Western blot analysis of DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion, was performed with the indicated antibodies. C Negative correlations were observed between the expression levels of HUWE1 and various mitochondrial proteins (VDAC1, TOMM20, TIMM22, NDUFB8, SDHB, UQCRC2, COX4, and ATP5A1) in CRC. D – F Measurement of mitochondrial ETC complex activities in DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion. G Measurement of ATP level in DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion. H Western blot analysis detected the expression of ABCC1, ABCB1, and ABCG2 in DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion. I DLD1, HT29, and HCT116 cells, with or without HUWE1 depletion, were incubated with 10 µM doxorubicin for 24 h. Cells were then stained with DAPI and observed by confocal microscopy for doxorubicin fluorescence. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: DLD1, HCT116, HT29, MCF7, HepG2, HeLa, and A549 cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: Western Blot, Expressing, Incubation, Staining, Confocal Microscopy, Fluorescence

Journal: Cell Death Discovery

Article Title: HUWE1 loss promotes stemness and drug resistance in CRC with dysregulated β-catenin destruction complex

doi: 10.1038/s41420-025-02731-2

Figure Lengend Snippet: DLD1 and HCT116 cells, with or without HUWE1 depletion, were transfected with the expression plasmid for resHUWE1, full-length APC, or both. A , B Western blot analysis of the indicated cells was performed using the specified antibodies. C – E Measurement of mitochondrial ETC complex activities in the indicated cells. F Measurement of ATP level in the indicated cells. G The IC50 of oxaliplatin, 5FU, and doxorubicin in the indicated cells was determined using the CCK-8 assay. H Apoptosis in the indicated cells was observed using flow cytometry analysis with Annexin V-FITC and PI dual staining after treatment with 50 µM oxaliplatin, 5FU, or doxorubicin for 48 h. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: DLD1, HCT116, HT29, MCF7, HepG2, HeLa, and A549 cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, CCK-8 Assay, Flow Cytometry, Staining

Journal: Cell Death Discovery

Article Title: HUWE1 loss promotes stemness and drug resistance in CRC with dysregulated β-catenin destruction complex

doi: 10.1038/s41420-025-02731-2

Figure Lengend Snippet: A Relationship between APC mutations and HUWE1 mRNA levels in 594 CRC samples. The data were extracted from the cBioPortal database ( https://www.cbioportal.org/ ). B Comparison of HUWE1 protein levels quantified by Western blot analysis in RKO, DLD1, HT29, and HCT116 cells. C Kaplan–Meier plot of overall survival for CRC patients with deleterious APC mutations, stratified by HUWE1 expression levels, analyzed using the log-rank test. D Schematic diagram summarizing the findings of this study. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: DLD1, HCT116, HT29, MCF7, HepG2, HeLa, and A549 cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea).

Techniques: Comparison, Western Blot, Expressing