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

Journal: Annals of Translational Medicine

Article Title: Downregulation of ubiquitin-specific protease 2 possesses prognostic and diagnostic value and promotes the clear cell renal cell carcinoma progression

doi: 10.21037/atm.2020.02.141

Figure Lengend Snippet: USP2 is significantly downregulated in ccRCC clinical samples and cell lines. (A,B,C,D) Expression profiles of USP2 mRNA in four renal statistics, including Jones, Gumz, Eroukhim and Lenburg renal statistics, were downloaded from Oncomine datasets and analyzed. (E,F) The mRNA levels in ccRCC downloaded from the TCGA-KIRC database consisting of 533 cases, including 72 paired cases. (G,H,I) Analysis of USP2 expression in clinical samples using qRT-PCR and IHC. (J,K) The protein expression of USP2 in ccRCC clinical samples and cell lines. ***P<0.001; ****P<0.0001.

Article Snippet: The second batch of sampled tissues, described above, was incubated with rabbit USP2 polyclonal antibody (ABclonal, A10399, 1:1,000) overnight at 4 °C, tissue sections washed with PBS then incubated with anti-rabbit secondary antibodies at room temperature for 2 hours.

Techniques: Expressing, Quantitative RT-PCR

Journal: Annals of Translational Medicine

Article Title: Downregulation of ubiquitin-specific protease 2 possesses prognostic and diagnostic value and promotes the clear cell renal cell carcinoma progression

doi: 10.21037/atm.2020.02.141

Figure Lengend Snippet: Correlation between USP2 mRNA expression and clinicopathological parameters of ccRCC patients.

Article Snippet: The second batch of sampled tissues, described above, was incubated with rabbit USP2 polyclonal antibody (ABclonal, A10399, 1:1,000) overnight at 4 °C, tissue sections washed with PBS then incubated with anti-rabbit secondary antibodies at room temperature for 2 hours.

Techniques: Expressing

Journal: Annals of Translational Medicine

Article Title: Downregulation of ubiquitin-specific protease 2 possesses prognostic and diagnostic value and promotes the clear cell renal cell carcinoma progression

doi: 10.21037/atm.2020.02.141

Figure Lengend Snippet: Low USP2 mRNA level is associated with various clinicopathological variables and negative prognosis in ccRCC patients. USP2 mRNA levels were compared in different clinicopathological variables: (A) male vs. female, (B) deceased vs. living, (C) G3 + G4 vs. G1 + G2, (D) T3 + T4 vs. T1 + T2, (E) TNM (III + IV) vs. TNM (I + II), (F) T stage, (G) TNM stage, (H) G stage, (I) non-metastasis vs. metastasis, (J) OS-good vs. OS-poor, (K) DFS-good vs. DFS-poor, and (L) non-recurrent vs. recurrent. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Article Snippet: The second batch of sampled tissues, described above, was incubated with rabbit USP2 polyclonal antibody (ABclonal, A10399, 1:1,000) overnight at 4 °C, tissue sections washed with PBS then incubated with anti-rabbit secondary antibodies at room temperature for 2 hours.

Techniques:

Journal: Annals of Translational Medicine

Article Title: Downregulation of ubiquitin-specific protease 2 possesses prognostic and diagnostic value and promotes the clear cell renal cell carcinoma progression

doi: 10.21037/atm.2020.02.141

Figure Lengend Snippet: Downregulation of USP2 correlated with overall survival and disease-free survival times of ccRCC patients. (A) A correlation between USP2 expression and OS times of 532 ccRCC patients analyzed by the Kaplan-Meier method. Overall survival analysis toward the expression of USP2 mRNA was performed in subgroups of ccRCC patients: (B) male, (C) age ≥60, (D) T1 + T2, (E) N0, (F) non-metastasis, (G) TNM I + TNM II, (H) G1 + G2, (I) G3 + G4. (J) The correlation between USP2 expression and DFS time for 434 ccRCC patients analyzed by Kaplan-Meier. DFs analysis toward the expression of USP2 mRNA was performed in subgroups of ccRCC patients: (K) male, (L) age ≥60 years, (M) non-metastasis, (N) T1 + T2, and (O) TNM I + TNM II.

Article Snippet: The second batch of sampled tissues, described above, was incubated with rabbit USP2 polyclonal antibody (ABclonal, A10399, 1:1,000) overnight at 4 °C, tissue sections washed with PBS then incubated with anti-rabbit secondary antibodies at room temperature for 2 hours.

Techniques: Expressing

Journal: Annals of Translational Medicine

Article Title: Downregulation of ubiquitin-specific protease 2 possesses prognostic and diagnostic value and promotes the clear cell renal cell carcinoma progression

doi: 10.21037/atm.2020.02.141

Figure Lengend Snippet: Downregulation of USP2 correlated with disease-free survival times of ccRCC patients. The correlation between USP2 expression and DFS time for ccRCC patients analyzed by Kaplan-Meier. DFs analysis toward the expression of USP2 mRNA was performed in subgroups of ccRCC patients: (A) M1, (B) T3 + T4, (C) Age <60 years, (D) N0, (E)N1, (F) TNM III+TNM IV, (G) G1 + G2, (H) G3 + G4, (I) female.

Article Snippet: The second batch of sampled tissues, described above, was incubated with rabbit USP2 polyclonal antibody (ABclonal, A10399, 1:1,000) overnight at 4 °C, tissue sections washed with PBS then incubated with anti-rabbit secondary antibodies at room temperature for 2 hours.

Techniques: Expressing

Journal: Annals of Translational Medicine

Article Title: Downregulation of ubiquitin-specific protease 2 possesses prognostic and diagnostic value and promotes the clear cell renal cell carcinoma progression

doi: 10.21037/atm.2020.02.141

Figure Lengend Snippet: Univariate and multivariate analysis of USP2 mRNA level and patient overall survival

Article Snippet: The second batch of sampled tissues, described above, was incubated with rabbit USP2 polyclonal antibody (ABclonal, A10399, 1:1,000) overnight at 4 °C, tissue sections washed with PBS then incubated with anti-rabbit secondary antibodies at room temperature for 2 hours.

Techniques:

Journal: Annals of Translational Medicine

Article Title: Downregulation of ubiquitin-specific protease 2 possesses prognostic and diagnostic value and promotes the clear cell renal cell carcinoma progression

doi: 10.21037/atm.2020.02.141

Figure Lengend Snippet: Univariate and multivariate analysis of USP2 mRNA level and patient disease-free survival

Article Snippet: The second batch of sampled tissues, described above, was incubated with rabbit USP2 polyclonal antibody (ABclonal, A10399, 1:1,000) overnight at 4 °C, tissue sections washed with PBS then incubated with anti-rabbit secondary antibodies at room temperature for 2 hours.

Techniques:

Journal: Annals of Translational Medicine

Article Title: Downregulation of ubiquitin-specific protease 2 possesses prognostic and diagnostic value and promotes the clear cell renal cell carcinoma progression

doi: 10.21037/atm.2020.02.141

Figure Lengend Snippet: Diagnostic value of USP2 mRNA expression in ccRCC patients. (A) ROC curve showing that USP2 could effectively distinguish ccRCC from para-cancer tissues. ROC curve analysis towards the expression of USP2 mRNA in subgroups of ccRCC patients against: (B) male vs. female, (C) TNM I + II vs. TNM III + IV, (D) G1 + G2 vs. G3 + G4, (E) T1 + T2 vs. T3 + T4, (F) living vs. deceased, (G) non-recurrent from recurrent, (H) OS-good from OS-poor, (I) DFS-good from DFS-poor.

Article Snippet: The second batch of sampled tissues, described above, was incubated with rabbit USP2 polyclonal antibody (ABclonal, A10399, 1:1,000) overnight at 4 °C, tissue sections washed with PBS then incubated with anti-rabbit secondary antibodies at room temperature for 2 hours.

Techniques: Diagnostic Assay, Expressing

Journal: Annals of Translational Medicine

Article Title: Downregulation of ubiquitin-specific protease 2 possesses prognostic and diagnostic value and promotes the clear cell renal cell carcinoma progression

doi: 10.21037/atm.2020.02.141

Figure Lengend Snippet: Overexpression of USP2 represses proliferation, migration, and invasion of ccRCC in vitro. (A,B,C) GSEA was performed to investigate the biological pathways involved in USP2 regulation based on the TCGA database. (D,E) USP2 mRNA and protein expressions were successfully overexpressed in A498 and caki-1 cells. (F,G) CCK-8 assays showing cell growth curves of A498 and CAKi-1 cells. (H,I) Representative images of migration and invasion assays performed using A498 and CAKi-1 cells; the number of cells was counted in ten randomly selected images from each group. **P<0.01; ***P<0.001; ****P<0.0001.

Article Snippet: The second batch of sampled tissues, described above, was incubated with rabbit USP2 polyclonal antibody (ABclonal, A10399, 1:1,000) overnight at 4 °C, tissue sections washed with PBS then incubated with anti-rabbit secondary antibodies at room temperature for 2 hours.

Techniques: Over Expression, Migration, In Vitro, CCK-8 Assay

Journal: Annals of Translational Medicine

Article Title: Downregulation of ubiquitin-specific protease 2 possesses prognostic and diagnostic value and promotes the clear cell renal cell carcinoma progression

doi: 10.21037/atm.2020.02.141

Figure Lengend Snippet: Potential value of USP2 in liquid biopsy. The expression of USP2 in blood and other body fluids was explored by analyzing the GeneCards and HPA RNA-seq normal tissues from PubMed.

Article Snippet: The second batch of sampled tissues, described above, was incubated with rabbit USP2 polyclonal antibody (ABclonal, A10399, 1:1,000) overnight at 4 °C, tissue sections washed with PBS then incubated with anti-rabbit secondary antibodies at room temperature for 2 hours.

Techniques: Expressing, RNA Sequencing