Journal: Cell Death Discovery

Article Title: Aberrant activation of a miR-101–UBE2D1 axis contributes to the advanced progression and chemotherapy sensitivity in human hepatocellular carcinoma

doi: 10.1038/s41420-024-02193-y

Figure Lengend Snippet: A Expression analysis of miR-101 in normal hepatic tissues (N) and LIHC tumor tissues (T) was conducted using data from the TCGA database and the GSE6857 dataset. B The differential expression of miR-101 between normal and paired LIHC tissues was analyzed using data from the TCGA database and the GSE6857 dataset. C Distribution of high, medium and low expression of miR-101 in HCC patients with different malignant degrees based on TCGA data. D The correlation between miR-101 expression levels and the overall survival of HCC patients was assessed using the Log-rank test. E The correlation between miR-101-responsive gene signatures (EED, EZH2, STMN1, JUNB) expression levels and the overall survival of HCC patients was derived from GEPIA. F Overlap of the predicted downstream targets of miR-101 from TargetScan and miRDB prediction databases. G The overlapping 595 predicted genes were subjected to KEGG analysis and enriched to the ubiquitin-mediated proteolysis pathway. The pathways were categorized based on the −log10 P value interval. H Fold-change differences with miR-101 low /miR-101 high based on TCGA data, UBE2D1 showed the most pronounced alteration in expression. I Correlation analysis of UBE2D1 and miR-101-responsive gene signatures in different tumors according to GEPIA. J A positive correlation was observed between the expression levels of UBE2D1 and EED, EZH2, STMN1, JUNB in LIHC tissues based on TCGA data. K A negative correlation was observed between the expression levels of UBE2D1 and miR-101 in LIHC tissues based on TCGA data. Statistical significance was determined by Student’s t -test. **** p < 0.0001.

Article Snippet: The membranes were blocked in 5% skim milk prepared in TBST buffer for 1 h and then incubated with primary antibodies against UBE2D1 (1:1000, Proteintech, 11677-1-AP), caspase 3 (1:1000, Cell Signaling Technology, #9662), γ-H2AX (1:2000, Abmart, M63324) and β-actin (1:2500, Cell Signaling Technology, #3700) overnight at 4 °C.

Techniques: Expressing, Quantitative Proteomics, Derivative Assay, Ubiquitin Proteomics

Journal: Cell Death Discovery

Article Title: Aberrant activation of a miR-101–UBE2D1 axis contributes to the advanced progression and chemotherapy sensitivity in human hepatocellular carcinoma

doi: 10.1038/s41420-024-02193-y

Figure Lengend Snippet: A Analysis of UBE2D1 expression in normal hepatic tissues (N) and LIHC tumor tissues (T) was conducted using data from the TCGA database and the GSE14520 dataset. B The differential expression of UBE2D1 between normal and paired LIHC tissues was analyzed by utilizing data from both the TCGA database and the GSE14520 dataset. C Distribution of high, medium and low expression of UBE2D1 in HCC patients with varying degrees of malignancy based on TCGA data. D Overall survival rate of HCC patients from the TCGA database was analyzed based on UBE2D1 mRNA levels using the Log-rank test. E Survival curve of 367 LIHC patients enrolled in TCGA database. Patients were classified into four groups based on the expression levels of miR-101 and UBE2D1 mRNA in their tumors. F The expression patterns of miR-101 and UBE2D1 mRNA in 367 LIHC samples and the statistical analysis of patient survival outcomes. Statistical significance was determined by Student’s t -test. **** p < 0.0001.

Article Snippet: The membranes were blocked in 5% skim milk prepared in TBST buffer for 1 h and then incubated with primary antibodies against UBE2D1 (1:1000, Proteintech, 11677-1-AP), caspase 3 (1:1000, Cell Signaling Technology, #9662), γ-H2AX (1:2000, Abmart, M63324) and β-actin (1:2500, Cell Signaling Technology, #3700) overnight at 4 °C.

Techniques: Expressing, Quantitative Proteomics

Journal: Cell Death Discovery

Article Title: Aberrant activation of a miR-101–UBE2D1 axis contributes to the advanced progression and chemotherapy sensitivity in human hepatocellular carcinoma

doi: 10.1038/s41420-024-02193-y

Figure Lengend Snippet: A Protein levels identification of shRNA-mediated UBE2D1 knockdown effect in SNU-739 and HCC-LM3 cells. B , C Cell viability was analyzed in stable UBE2D1-knockdown cell lines following treatment of 0.5 μM cDDP for 0, 24, 48 and 72 h respectively and treatment of 0.5 μM 5Fu for 0, 24, 48 and 72 h respectively by CCK8 assay. D , E Cell proliferation ability detection of stable UBE2D1-knockdown cell lines after treatment with 0.5 μM cDDP or 0.5 μM 5Fu by plate clone formation assay. F A western blotting analysis of cleaved caspase 3 protein levels in UBE2D1-knockdown cells with or without the cDDP (50 μM, 24 h) or 5Fu (50 μM, 48 h) treatment. G Apoptosis rate detection of UBE2D1-knockdown cells after treatment with cDDP or 5Fu (5 μM, 24 h) by flow cytometry. All experiments were performed three times. Statistical significance was determined by Student’s t -test. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: The membranes were blocked in 5% skim milk prepared in TBST buffer for 1 h and then incubated with primary antibodies against UBE2D1 (1:1000, Proteintech, 11677-1-AP), caspase 3 (1:1000, Cell Signaling Technology, #9662), γ-H2AX (1:2000, Abmart, M63324) and β-actin (1:2500, Cell Signaling Technology, #3700) overnight at 4 °C.

Techniques: shRNA, Knockdown, CCK-8 Assay, Tube Formation Assay, Western Blot, Flow Cytometry

Journal: Cell Death Discovery

Article Title: Aberrant activation of a miR-101–UBE2D1 axis contributes to the advanced progression and chemotherapy sensitivity in human hepatocellular carcinoma

doi: 10.1038/s41420-024-02193-y

Figure Lengend Snippet: A Protein levels identification of UBE2D1 overexpression effect in SNU-739 and HCC-LM3 cells. B , C Cell viability was assessed in stable UBE2D1-overexpression cells following treatment of 0.5 μM cDDP for 0, 24, 48, and 72 h respectively and treatment of 0.5 μM 5Fu for 0, 24, 48, 72 h respectively by CCK8 assay. D , E The cell proliferation ability of stable UBE2D1-overexpression cells was assessed after treatment with 0.5 μM cDDP or 0.5 μM 5Fu using plate clone formation assay. F A western blotting analysis of cleaved caspase 3 protein levels in stable UBE2D1-overexpression cells with or without the cDDP (50 μM, 24 h) or 5Fu (50 μM, 48 h) treatment. G Apoptosis rate of stable UBE2D1-overexpression cells was assessed by flow cytometry after treatment with cDDP or 5Fu (5 μM, 24 h). All experiments were performed three times. Statistical significance was determined by Student’s t -test. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: The membranes were blocked in 5% skim milk prepared in TBST buffer for 1 h and then incubated with primary antibodies against UBE2D1 (1:1000, Proteintech, 11677-1-AP), caspase 3 (1:1000, Cell Signaling Technology, #9662), γ-H2AX (1:2000, Abmart, M63324) and β-actin (1:2500, Cell Signaling Technology, #3700) overnight at 4 °C.

Techniques: Over Expression, CCK-8 Assay, Tube Formation Assay, Western Blot, Flow Cytometry

Journal: Cell Death Discovery

Article Title: Aberrant activation of a miR-101–UBE2D1 axis contributes to the advanced progression and chemotherapy sensitivity in human hepatocellular carcinoma

doi: 10.1038/s41420-024-02193-y

Figure Lengend Snippet: A Western blotting analysis of γ-H2AX protein levels in HCC cells following stable knockdown of UBE2D1, with or without treatment of cDDP or 5Fu (10 μM, 2 h). B Representative images and quantification of γ-H2AX foci in stable UBE2D1-knockdown cells following treatment with or without cDDP or 5Fu (10 μM, 2 h). γ-H2AX accumulates at DNA double-strand break sites (scale bar = 10 μm). The statistical scatter points represent the number of γ-H2AX foci in each cell, which were counted by Image J software. C The comet assay was employed to evaluate the extent of DNA damage in stable UBE2D1-knockdown cells treated with or without cDDP or 5Fu (10 μM, 12 h). The head of the comet as a spherical mass of undamaged DNA, while the damaged DNA (DNA loops around strand breaks) emanates from the head in the form of a tail (scale bar = 150 μm). D Western blotting analysis of γ-H2AX protein levels in HCC cells following stable overexpression of UBE2D1, with or without the cDDP or 5Fu treatment (10 μM, 2 h). E Representative images and quantification of γ-H2AX foci in stable UBE2D1-overexpression cells following treatment with or without cDDP or 5Fu (10 μM, 2 h) (scale bar = 10 μm). F Comet assay was performed on stable UBE2D1-overexpression cells, with or without treatment of cDDP or 5Fu (10 μM, 12 h) (scale bar = 150 μm). All experiments were performed three times. Statistical significance was determined by Student’s t -test. **** p < 0.0001, *** p < 0.001, ** p < 0.01.

Article Snippet: The membranes were blocked in 5% skim milk prepared in TBST buffer for 1 h and then incubated with primary antibodies against UBE2D1 (1:1000, Proteintech, 11677-1-AP), caspase 3 (1:1000, Cell Signaling Technology, #9662), γ-H2AX (1:2000, Abmart, M63324) and β-actin (1:2500, Cell Signaling Technology, #3700) overnight at 4 °C.

Techniques: Western Blot, Knockdown, Software, Single Cell Gel Electrophoresis, Over Expression

Journal: Cell Death Discovery

Article Title: Aberrant activation of a miR-101–UBE2D1 axis contributes to the advanced progression and chemotherapy sensitivity in human hepatocellular carcinoma

doi: 10.1038/s41420-024-02193-y

Figure Lengend Snippet: A The predicted binding sites between the wild-type (WT) and mutant (MUT) 3ʹUTR of UBE2D1 and miR-101. B WT UBE2D1 3ʹUTR and MUT UBE2D1 3ʹUTR firefly luciferase activities were measured in HEK293T cells transfected with NC mimics or miR-101 mimics. C , D Western blotting analysis of UBE2D1 expression in SNU-739 and HCC-LM3 cells transfected with miR-101 mimics or miR-101 inhibitors. E , F The miR-101 and UBE2D1 transcript levels were quantified by qRT-PCR in cells transfected with miR-101 mimics or miR-101 inhibitors. Graph represents miR-101 and UBE2D1 mRNA levels, expressed as fold changes relative to the control. All experiments were performed three times. Statistical significance was determined by Student’s t -test. **** p < 0.0001, *** p < 0.001, * p < 0.05, ns nonsignificant.

Article Snippet: The membranes were blocked in 5% skim milk prepared in TBST buffer for 1 h and then incubated with primary antibodies against UBE2D1 (1:1000, Proteintech, 11677-1-AP), caspase 3 (1:1000, Cell Signaling Technology, #9662), γ-H2AX (1:2000, Abmart, M63324) and β-actin (1:2500, Cell Signaling Technology, #3700) overnight at 4 °C.

Techniques: Binding Assay, Mutagenesis, Luciferase, Transfection, Western Blot, Expressing, Quantitative RT-PCR, Control

Journal: Cell Death Discovery

Article Title: Aberrant activation of a miR-101–UBE2D1 axis contributes to the advanced progression and chemotherapy sensitivity in human hepatocellular carcinoma

doi: 10.1038/s41420-024-02193-y

Figure Lengend Snippet: A Western blotting analysis of UBE2D1 expression changes in SNU-739 and HCC-LM3 cells following stable transfection of miR-101 and rescue expression of UBE2D1 on this basis. B The miR-101 and UBE2D1 transcript levels were quantified by qRT-PCR in miR-101 overexpression cells and rescued cells. C , D Cell proliferation of HCC cells was analyzed in the rescue experiments following treatment of 0.5 μM cDDP or 0.5 μM 5Fu for 0, 24, 48 and 72 h respectively by CCK8 assay. E , F The cell proliferation of HCC cells was analyzed in the rescue experiments after treatment with 0.5 μM cDDP or 0.5 μM 5Fu using plate clone formation assay. G Cleaved caspase 3 expression levels were analyzed using western blotting in the rescue experiments performed on SNU-739 and HCC-LM3 cells, with or without administration of cDDP (50 μM, 24 h) or 5Fu (50 μM, 48 h). H Cell apoptosis rate was detected by flow cytometry in the rescue experiments with the cDDP or 5Fu treatment (5 μM, 24 h). I , J Detection of γ-H2AX DNA damage foci in the rescue experiments of SNU-739 and HCC-LM3 cells by western blotting and immunofluorescent staining (scale bar = 10 μm). The statistical scatter points represent the quantification of γ-H2AX foci per cell. K Comet assay was performed on the rescue experiments with or without administration of cDDP or 5Fu (10 μM, 12 h) (scale bar = 150 μm). All experiments were performed three times. Statistical significance was determined by Student’s t -test. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: The membranes were blocked in 5% skim milk prepared in TBST buffer for 1 h and then incubated with primary antibodies against UBE2D1 (1:1000, Proteintech, 11677-1-AP), caspase 3 (1:1000, Cell Signaling Technology, #9662), γ-H2AX (1:2000, Abmart, M63324) and β-actin (1:2500, Cell Signaling Technology, #3700) overnight at 4 °C.

Techniques: Western Blot, Expressing, Stable Transfection, Quantitative RT-PCR, Over Expression, CCK-8 Assay, Tube Formation Assay, Flow Cytometry, Staining, Single Cell Gel Electrophoresis

Journal: Cell Death Discovery

Article Title: Aberrant activation of a miR-101–UBE2D1 axis contributes to the advanced progression and chemotherapy sensitivity in human hepatocellular carcinoma

doi: 10.1038/s41420-024-02193-y

Figure Lengend Snippet: A Representative tumor images in nude mice after the injection of SNU-739 cells that stably expressing UBE2D1-shRNA and control scramble shRNA, with or without the cDDP treatment (2 mg/kg/3 days). B , C Growth curves of xenograft tumors in nude mice and quantification of tumor weight. The initiation time of drug administration is recorded as 0 d. D–F Representative images of γ-H2AX, Ki67 and tunel staining in tumor samples. On the right is the result of statistical analysis on positive signals using Image J Profiler and GraphPad Prism (scale bar = 20 μm). All experiments were performed three times. Statistical significance was determined by Student’s t -test. *** p < 0.001, * p < 0.05, ns nonsignificant.

Article Snippet: The membranes were blocked in 5% skim milk prepared in TBST buffer for 1 h and then incubated with primary antibodies against UBE2D1 (1:1000, Proteintech, 11677-1-AP), caspase 3 (1:1000, Cell Signaling Technology, #9662), γ-H2AX (1:2000, Abmart, M63324) and β-actin (1:2500, Cell Signaling Technology, #3700) overnight at 4 °C.

Techniques: Injection, Stable Transfection, Expressing, shRNA, Control, TUNEL Assay, Staining