Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: The association between USP2 expression and clinicopathological parameters in bladder cancer patients.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: Abnormally elevated expression of USP2 in BLCA tissues and cell lines. (A) RT-qPCR analysis of USP2 level in tumor tissues and adjacent normal tissues of 46 BLCA patients. (B) The protein level of USP2 was examined in 4 BLCA patients. (C) RT-qPCR was used to test USP2 expression in BLCA tissues. ΔCTN represented comparative Ct in normal tissues. ΔCTC was comparative Ct in tumor tissues. (D) Kaplan-Meier survival analysis of the relationship between USP2 level and overall survival of BLCA patients. (E, F) RT-qPCR and western blot analysis of USP2 level in BLCA cell lines and normal bladder epithelial cells SV-HUC-1. Values are mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: Depletion of USP2 weakened cell proliferation, migration and invasion of BLCA cells. (A, B) USP2 expression was extremely decreased in 5637 and J82 cells transfected with sh-USP2#1 or sh-USP2#2. (C) CCK-8 assay displayed cell growth curves in 5637 and J82 cells. (D) Representative images and quantification of EdU assay were performed in BLCA cells. (E) The migration of BLCA cells was assessed using the wound healing assay. (F) Transwell assay was used to test the capacity of cell invasion in BLCA cells. Results expressed as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Migration, Expressing, Transfection, CCK-8 Assay, EdU Assay, Wound Healing Assay, Transwell Assay

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: EZH2 was increased in BLCA and correlated with USP2 level. (A) RT-qPCR analysis of EZH2 level in tumor tissues and adjacent normal tissues of 46 BLCA patients. (B) RT-qPCR was used to test EZH2 expression in BLCA tissues. (C) Kaplan-Meier survival analysis of the relationship between EZH2 level and overall survival of BLCA patients. (D) The correlation between USP2 and EZH2 levels in BLCA tissues was assessed using Spearman analysis. (E) RT-qPCR analysis of EZH2 level in BLCA cell lines and normal bladder epithelial cells. (F) RT-qPCR detection of EZH2 expression in 5637 and J82 cells transfected with sh-USP2. (G) Western blot analysis of EZH2 and H3K27me3 levels in 5637 and J82 cells transfected with sh-USP2. Data are represented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: USP2 stabilized EZH2 by inhibiting its ubiquitination and degradation in BLCA cells. (A) 5637 and J82 cell lysates were immunoprecipitated using antibodies against USP2 or EZH2, followed by immunoblotting with the indicated antibodies. (B) BLCA cells were transfected with His-USP2 and HA-EZH2. Cell lysates were immunoprecipitated using anti-His magnetic beads, and immunoblotted with His and HA antibodies. (C) Cells overexpressing USP2 were treated with 50 ng/ml Cycloheximide (CHX) for 3, 6, and 9 h. Western blot was used to detect EZH2 expression. (D) Western blot was used to detect the expressions of USP2 and EZH2 in cells transfected with sh-NC or sh-USP2 with/without proteasomal inhibitor MG132 (20 μM) for 5 h. (E) EZH2 in BLCA cells transfected with His-USP2 or sh-USP2 was immunoprecipitated to detect ubiquitylation. Data are represented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Transfection, Magnetic Beads, Expressing

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: Overexpressed EZH2 abolished USP2 silencing-mediated biological functions on BLCA cells. We knocked down the level of USP2 and overexpressed EZH2 expression in 5637 cells. (A) The CCK-8 assay displayed cell growth curves in 5637 cells. (B) Representative images and quantification of EdU assay in 5637 cells. (C) Transwell detection of invasion in 5637 cells. (D) Western blot analysis of USP2 and EZH2 protein levels. Values were expressed as mean ± SD of three separate determinations. ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, CCK-8 Assay, EdU Assay, Western Blot

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: USP2 epigenetically repressed SOX1 expression via EZH2 stabilization. RT-qPCR was used to detect the levels of candidate gene in BLCA cells transfected with sh-USP2 (A) or si-EZH2 (B). (C, D) ChIP assay was performed to assess the occupancy of EZH2 and H3K27me3 in the SOX1 promoter region in cells with sh-NC or sh-USP2. (E, F) RT-qPCR and western blot analysis of EZH2 and SOX1 expression in BLCA cells transfected with sh-USP2 and/or pcDNA3.1-EZH2. Error bars stand for the mean ± SD of at least triplicate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot

Journal: Translational Oncology

Article Title: The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression

doi: 10.1016/j.tranon.2024.102104

Figure Lengend Snippet: The USP2-EZH2-SOX1 axis in tumor growth of BLCA in vivo . The 5637 cells stably expressing sh-USP2 or EZH2 were separately injected into the flanks of BALB/c nude mice to establish a xenograft tumor model. (A) Representative photographs of tumors from the xenograft nude mice. (B) Tumor weight in the three groups. (C) Tumor volumes were determined on the indicated days. (D) IHC analysis detected the expression of SOX1 in tumor tissues. (E) Schematic graph illustrating the role of USP2 in regulating the progression of BLCA. The results are representative of three independent experiments. All data are represented as mean ± SD. ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: In Vivo, Stable Transfection, Expressing, Injection

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: USP2 protein expression levels were reduced in HCC-T tissues from HCC patients. (A) Representative total USP2 protein staining with goat anti-human USP2 IgG in the tissues of human normal liver (n=76), HCC-NT (n=38) and HCC-T (n=115) by IHC. As negative controls, human normal liver tissues (n=6) were also stained with goat pre-immune serum. (B) Quantification of the USP2 protein levels were performed with ImageJ. (C) Representative USP2 protein staining in four paired HCC-T and HCC-NT tissues (n=10) by IHC. (D) USP2 protein levels in ten paired HCC-NT and HCC-T tissues as groups as well as (E) individual pairs. **P<0.01 and ***P<0.001 in students’ t-test for pair-wise comparison or one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Expressing, Staining, Comparison

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: USP2 mRNA expression levels were decreased in HCC-T tissues from HCC patients. (A) The expression levels of USP2 mRNA in human normal liver (n=12), HCC-NT (n=8) and HCC-T (n=21) detected by real-time PCR. (B) The expression levels of USP2 mRNA in paired HCC-NT and HCC-T tissues as groups as well as (C) individual pairs (n=8). *P<0.05 and **P<0.01 in students’ t-test for pair-wise comparison or one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Comparison

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: Dysregulation of Usp2 mRNA expression in HCC-T tissues from HCC mice. A. The expression levels of Usp2 mRNA in mouse normal liver (n=16), HCC-NT (n=15) and HCC-T (n=15) samples. B. The expression levels of USP2 mRNA in paired HCC-T and HCC-NT tissues. **P<0.01 and ***P<0.001 in students’ t-test for pair-wise comparison or one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Expressing, Comparison

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: USP2b is the predominant USP2 isoform in the liver. (A) The relative mRNA expression levels of USP2a, USP2b and USP2c in normal liver tissues from human (n=12) and (B) mice (n=16). **P<0.01 and ***P<0.001 in one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: USP2b mRNA expression levels were markedly reduced in HCC-T tissues from HCC patients. (A) The expression levels of USP2 mRNA in human normal liver (n=12), HCC-NT (n=8) and HCC-T (n=21) detected by real-time PCR. (B) USP2b protein detected by Western blot in normal liver (n=8), HCC-NT (n=8) and HCC-T tissues (n=8). (C) Quantification of the USP2b protein levels in (B) by ImageJ. (D) The expression levels of USP2 mRNA in paired HCC-NT and HCC-T tissues as groups as well as (E) individual pairs (n=8). ***P<0.001 in students’ t-test for pair-wise comparison or one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Comparison

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: USP2b promoted cell proliferation. (A) HepG2 and (B) Huh 7 cells were transfected with USP2b or pcDNA vector, followed by monitoring cell growth for a period of 48 h by the MTS assays. (C) HepG2 and (D) Huh 7 cells were treated with increasing concentrations of USP2-specific inhibitor ML364 for 48 h, followed by monitoring cell proliferation by MTS assays. (E) HepG2 and (F) Huh 7 cells were transfected with USP2b or pcDNA vector in the presence or absence of USP2 inhibitor ML364 for 48 h, followed by monitoring cell proliferation by MTS assays. *P<0.05, **P<0.01, ***P<0.001 in students’ t-test for pair-wise comparison or one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Transfection, Plasmid Preparation, Comparison

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: Knockout of USP2 significantly reduced cell proliferation. (A) Relative proliferation of parental HepG2 and USP2-KO (3A) and (B) USP2-KO (3E) cells over a period of 96 h. (C) USP2-KO (3A) and (D) USP2-KO (3E) cells were transfected with USP2b or pcDNA vector, followed by monitoring the cell proliferation over a period of 48 h. *P<0.05, **P<0.01 and ***P<0.001 in students’ t-test for pair-wise comparison.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Knock-Out, Transfection, Plasmid Preparation, Comparison

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: Identification of USP2-regulated target proteins with proteomic profiling. (A) Quantitative analysis of the proteomics dataset obtained from parental HepG2 (n=3 duplicates) and USP2-KO (3A) cells (n=3 duplicates) and (B) from HepG2 wt and USP2-KO (3E) cells (n=3 duplicates). Volcano plot shows the fold-change (1.5 fold up or down) versus p-value of protein abundance for the total of 4861 proteins identified. Red and green circles represent the significant (q<0.05) up- and down-regulated proteins in USP2-KO (3A) or USP2-KO (3E) compared with the parental HepG2 cells. (C) Overlap of the proteins differentially expressed in USP2-KO (3A) and USP2-KO (3E) cells when compared with the parental HepG2 cells. (D) The protein enrichment analysis of the overlapped altered proteins by KEGG and ShinyGo. (E) Overlap of upregulated proteins differentially expressed in USP2-KO (3A) and USP2-KO (3E) cells when compared with HepG2 cells. (F) The protein enrichment analysis of the overlapped upregulated proteins by KEGG and ShinyGo. (G) Overlap of downregulated proteins differentially expressed in USP2-KO (3A) and USP2-KO (3E) cells when compared with parental HepG2 cells. (H) The protein enrichment analysis of the overlapped downregulated proteins by KEGG and ShinyGo. (I) The heat map analysis of the USP2 regulated target proteins associated with cell proliferation, apoptosis, and tumorigenesis in the parental HepG2, USP2-KO (3A) and USP2-KO (3E) cells.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Quantitative Proteomics, Protein Enrichment

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: Up- and down-regulated proteins in USP2-KO (3A) and USP2-KO (3E) cells when compared to parental HepG2 cells

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques:

Journal: American Journal of Cancer Research

Article Title: Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma

doi:

Figure Lengend Snippet: Confirmation of USP2-regulated target proteins by Western blotting. (A) Detection and quantification of USP2-regulated target protein STK4, (B) DPP4, (C) FABP1 and (D) EGFR in parental HepG2, USP2-KO (3A) and USP2-KO (3E) cells. ***P<0.001 with one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Article Snippet: Liver tissue slides were blocked with 10% normal donkey serum (Abcam) followed by incubation with goat anti-mouse USP2 IgG (AF5804, R & D Systems) or pre-immunized serum (Biomatik).

Techniques: Western Blot