Journal: The Journal of Physiology

Article Title: Statin myalgia is not associated with reduced muscle strength, mass or protein turnover in older male volunteers, but is allied with a slowing of time to peak power output, insulin resistance and differential muscle mRNA expression

doi: 10.1113/jphysiol.2014.285577

Figure Lengend Snippet: List genes used on low density RT‐PCR array microfludic cards (Applied Biosystems Inc., Foster City, CA, USA)

Article Snippet: , USP19 , Ubiquitin specific peptidase 19 , Hs00324123_m1.

Techniques: Binding Assay, Phospho-proteomics, Ubiquitin Proteomics

Journal: OncoTargets and Therapy

Article Title:

USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/ott.s240543

Figure Lengend Snippet: Figure 1 Relative expression of USP19 in GC cell lines and GES1. (A) Differential expression of USP19 mRNA in gastric cell lines by real-time PCR assay. (B) The differential expression of USP19 protein in gastric cell lines by WB analysis. (C) Differential expression and distribution of USP19 protein in gastric cell lines by confocal analysis, Scale bar=20 μm. (D) Relative expression of USP19 in paired 212 GC samples and ANTs by IHC staining and analysis by Chi-square test. (E) USP19 protein levels in GC tissues and ANTs were analyzed by IHC staining (200× magnification). High or low expression of USP19 protein in ANTs (a and b); high or low expression of USP19 protein in intestinal-type GC (c and d); high or low expression of USP19 protein in diffuse-type GC (e and f), Scale bar=200 μm.

Article Snippet: The slides were incubated at 4°C overnight with mouse anti-human USP19 (dilution 1:200; Proteintech, Chicago, IL) for human samples, and with USP19 antibody (dilution 1:200), rabbit anti-human MMP2 and MMP9, cleaved caspase-3 (dilution 1:100, respectively; Cell Signaling Technology, Danvers, MA) for xenograft tissues.

Techniques: Expressing, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Immunohistochemistry

Journal: OncoTargets and Therapy

Article Title:

USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/ott.s240543

Figure Lengend Snippet: Figure 2 Kaplan–Meier survival curves of GC patients according to the clinical features and expression of USP19. (A) Patients with high-level USP19 expression showed poor survival. (B) TNM stage was a promising indicator of the prognosis of GC patients. (C) Patients with intestinal-type GC had better survival than those with diffuse-type GC. (D) Low-level USP19 expression predicted better survival in patients with TNM stage III. (E) Low-level USP19 expression predicted better survival in patients with diffuse-type GC. (F) Low-level USP19 expression predicted better survival in patients with intestinal-type GC.

Article Snippet: The slides were incubated at 4°C overnight with mouse anti-human USP19 (dilution 1:200; Proteintech, Chicago, IL) for human samples, and with USP19 antibody (dilution 1:200), rabbit anti-human MMP2 and MMP9, cleaved caspase-3 (dilution 1:100, respectively; Cell Signaling Technology, Danvers, MA) for xenograft tissues.

Techniques: Expressing

Journal: OncoTargets and Therapy

Article Title:

USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/ott.s240543

Figure Lengend Snippet: Figure 3 Functional roles of USP19 in cell models. (A) Knockdown of USP19 inhibited SGC7901 cell growth, as determined by MTT assay. Bars: SD; **P<0.01. (B) Knockdown of USP19 decreased the level of MMP2 and MMP9 and increased the level of the apoptotic-related protein, cleaved caspase-3, in SGC7901 cells. (C) Knockdown of USP19 inhibited colony formation in SGC7901 cells. The data are shown as Mean±SD (n=3). **P<0.01. (D) Ectopic expression of USP19 promoted GES1 cell growth, as shown by MTT assay. Bars: SD; **P<0.01. (E) Overexpression of USP19 increased the protein level of MMP2 and MMP9 and decreased the level of the apoptotic-related protein, cleaved caspase-3. (F) Ectopic expression of USP19 increased colony formation in GES1 cells. The data are shown as Mean±SD (n=3). **P<0.01. All WB experiments were repeated three times.

Article Snippet: The slides were incubated at 4°C overnight with mouse anti-human USP19 (dilution 1:200; Proteintech, Chicago, IL) for human samples, and with USP19 antibody (dilution 1:200), rabbit anti-human MMP2 and MMP9, cleaved caspase-3 (dilution 1:100, respectively; Cell Signaling Technology, Danvers, MA) for xenograft tissues.

Techniques: Functional Assay, Knockdown, MTT Assay, Expressing, Over Expression

Journal: OncoTargets and Therapy

Article Title:

USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/ott.s240543

Figure Lengend Snippet: Figure 4 Effect of USP19 on cell invasion and migration and MMP2/MMP9 enzyme activity. (A) Knockdown of USP19 reduced SGC7901 cell invasion. Images were acquired at the indicated time (200× magnification). Data are shown as Mean±SD (n=5). **P<0.01. (B) Knockdown of USP19 reduced the migration of SGC7901 cells, as determined by wound healing assay at 0, 12, 24, and 36 h. Images were acquired at the indicated time (100× magnification). Data are shown as Mean±SD (n=3). **P<0.01. (C) SGC7901 cells were transfected with ShUSP19 or vector for 36 h and then subjected to gelatin zymography to analyze the activity of MMP2 and MMP9. (D) Ectopic expression of USP19 enhanced invasion of GES1 cells. Images were acquired at the indicated time (200× magnification). Data are shown as Mean±SD (n=5). **P<0.01. (E) Ectopic expression of USP19 increased the migration of GES1 cells, as determined by wound healing assay at 0, 12, 24, and 36 h. Images were acquired at the indicated time (100× magnification). Data are shown as Mean±SD (n=3). **P<0.01. (F) GES1 cells were transfected with USP19-overexpressing plasmid or control vector for 36 h and then subjected to gelatin zymography to analyze the activity of MMP2 and MMP9.

Article Snippet: The slides were incubated at 4°C overnight with mouse anti-human USP19 (dilution 1:200; Proteintech, Chicago, IL) for human samples, and with USP19 antibody (dilution 1:200), rabbit anti-human MMP2 and MMP9, cleaved caspase-3 (dilution 1:100, respectively; Cell Signaling Technology, Danvers, MA) for xenograft tissues.

Techniques: Migration, Activity Assay, Knockdown, Wound Healing Assay, Transfection, Plasmid Preparation, Zymography, Expressing, Control

Journal: OncoTargets and Therapy

Article Title:

USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/ott.s240543

Figure Lengend Snippet: Figure 5 Knockdown of USP19 reduced gastric carcinogenesis in animal models. Knockdown of USP19 inhibited tumor growth. Five nude mice were injected subcutaneously with 1 × 107 cells/mouse for each of the indicated stable cell lines of SGC7901-ShUSP19 or SGC7901-Vector, respectively. Results were presented as tumor volume at the different time points (C) and isolated xenografts at day 24 (A) with no change in body weight for each group (B). **P<0.01. (D) Tumors were isolated, fixed, and subjected to IHC assay (400× magnification).

Article Snippet: The slides were incubated at 4°C overnight with mouse anti-human USP19 (dilution 1:200; Proteintech, Chicago, IL) for human samples, and with USP19 antibody (dilution 1:200), rabbit anti-human MMP2 and MMP9, cleaved caspase-3 (dilution 1:100, respectively; Cell Signaling Technology, Danvers, MA) for xenograft tissues.

Techniques: Knockdown, Injection, Stable Transfection, Plasmid Preparation, Isolation

Journal: OncoTargets and Therapy

Article Title:

USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/ott.s240543

Figure Lengend Snippet: Figure 6 Prognostic roles of USP19 was determined in www.kmplot.com and correlation between mRNA levels of USP19 and MMP2/MMP9 in GC dataset from GEPIA Website (http://gepia2.cancer-pku.cn). Overall survival curves were plotted for all GC patients (n=876) with (A) different levels of USP19 expression; (B) Lauren’s classification; (C and D) HER2 status, (E) Different treatments, 5-FU based chemical therapy, (F) Surgery alone. From these data, low level of USP19 expression was correlated with better OS. The mRNA level of USP19 expression was positive association with MMP2/MMP9 expression. (G)P<0.001 correlation between MMP9 and USP19; (H) P=0.062, correlation between MMP2 and USP19. Especially, the moderate correlation was performed between USP19 and MMP9 expression (Spearman’s R=0.24).

Article Snippet: The slides were incubated at 4°C overnight with mouse anti-human USP19 (dilution 1:200; Proteintech, Chicago, IL) for human samples, and with USP19 antibody (dilution 1:200), rabbit anti-human MMP2 and MMP9, cleaved caspase-3 (dilution 1:100, respectively; Cell Signaling Technology, Danvers, MA) for xenograft tissues.

Techniques: Expressing

Journal: bioRxiv

Article Title: Common molecular mechanisms underlie the transfer of alpha-synuclein, Tau and huntingtin and modulate spontaneous activity in neuronal cells

doi: 10.1101/2021.07.18.452825

Figure Lengend Snippet: (A) Immunoblots showing protein levels in cell lysates and released to the cell media of cells expressing the different proteins. HEK cell stably expressing 25QHtt-EGFP, 103QHtt-EGFP, aSyn-EGFP and EGFP-Tau were transfected with USP19 or with the catalytic inactive form USP19 C506S. Quantifications were normalized to total protein levels using MemCode. (B) LDH measurements confirm the absence of cell toxicity and cell death in the experiments. (C) Tau is more strongly internalized by naïve cells. Percentage of EGFP positive cells after incubation with media from cells co-expressing 25QHtt-EGFP, 103QHtt-EGFP, aSyn-EGFP or EGFP-Tau together with USP19 or USP19 C506S for 24 hours. Cell counting was performed using flow cytometry. Data from at least three independent experiments for each condition. Significant differences were assessed by one-way ANOVA followed by multiple comparisons with significance between groups corrected by Bonferroni procedure. Differences were considered to be significant for values of p<0.05 and are expressed as mean ± SD, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: USP19 plasmids were kindly provided by Prof. Dr. Yihong Ye through Addgene , and Htt plasmids were kindly provided by NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research.

Techniques: Western Blot, Expressing, Stable Transfection, Transfection, Incubation, Cell Counting, Flow Cytometry

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: ( A ) Workflow used to select a candidate regulatory gene. MDAMB231 cells were stably transduced with control empty vector shRNA (control) or two different shRNAs (#1 & #2) targeting USP19. ( B ) Efficiencies of shRNA-mediated knockdown were confirmed by RT-PCR (top, n= 5, one-way ANOVA, Dunnett’s multiple comparison test. shRNA#1 p= 0.0048 and shRNA#2 p= 0.0006) and Western Blotting (middle and bottom, n= 5, one-way ANOVA, Dunnett’s multiple comparison test. shRNA#1 p= 0.0064 and shRNA#2 p= 0.0003). ( C ) Crystal violet (CV) staining was used to determine cell growth over time. Cells were seeded onto wells and allowed to attach. At the indicated time points, cells were fixed and then stained at the end of the experiment. The graph on the top shows the mean relative CV absorbance every 24 hours. Doubling time was calculated for control and USP19 silenced cell lines on the bottom (n≥ 3, Kruskal-Wallis, Dunn’s multiple comparison test. shRNA#1 p> 0.9999 and shRNA#2 p> 0.9999). Migratory potential was evaluated by two different experiments. ( D ) Transwell assay: After 24 hours of incubation, USP19-depleted cells were stained for microscopic examination and the number of migratory cells was compared to control cells. The graph shows the number of migratory cells per transwell membrane (n= 4, one-way ANOVA, Dunnett’s multiple comparison test. shRNA#1 p= 0.0187 and shRNA#2 p= 0.0001). ( E ) Wound healing assays: scratching with a pipette tip made a gap on a monolayer of the different cell cultures, and time-lapse imaging monitored the number of migrating cells across the border. After 8 hours, cells exhibited different levels of migration. The graph on the left shows the gap covered area (mm2) after 8 hours (n= 16, one-way ANOVA, Dunnett’s multiple comparison test. shRNA#1 p< 0.0001 and shRNA#2 p< 0.0001) and the images on the right show representative areas in a wound healing experiment at the indicated time points. Scale bar= 100 μm.

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: Stable Transfection, Transduction, Control, Plasmid Preparation, shRNA, Knockdown, Reverse Transcription Polymerase Chain Reaction, Comparison, Western Blot, Staining, Transwell Assay, Incubation, Membrane, Transferring, Imaging, Migration

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: An area-based analysis of proliferation rate was used to determine cell growth over time. Cells were seeded onto wells, allowed to attach and imaged at the indicated time points. Left: relative mean occupied area at each time point ± S.E., and right: doubling time calculated for control and USP19 silenced cell lines (n= 9, one-way ANOVA, Dunnett’s multiple comparison test. shRNA#1 p= 0.6284 and shRNA#2 p= 0.2123).

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: Control, Comparison, shRNA

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: We generated MDAMB231 cells expressing GFP tagged-H2B and then stably transduced them with control vector or shRNAs # 1 and # 2 directed against USP19. For the wound healing assays, a scratch was made with a pipette tip on a monolayer of the different cell cultures, and time-lapse imaging monitored the number of migrating cells across the border. ( A ) Representative images of a wound healing assay at time= 0 hours. Cells circled in magenta were considered for the analysis. Data corresponding to circled cells in the gap area were manually excluded from the analysis. Data was collected from three independent experiments for each cell line. Scale bar= 100 μm. ( B ) The graph shows the gap covered area (mm 2 ) at the indicated time. ( C ) Cells mean speed from each wound-edge was calculated throughout the length of the experiment. (n=6, one-way ANOVA, Dunnett’s multiple comparison test. shRNA#1 p< 0.0001 and shRNA#2 p= 0.0028). ( D ) Mean accumulated track displacement (total displacement) for cells in each wound-edge was calculated until the end of the experiment (n=6, one-way ANOVA, Dunnett’s multiple comparison test. shRNA#1 p< 0.0001 and shRNA#2 p= 0.0059). ( E ) Persistence was calculated as the Euclidean/Accumulated track displacement ratio for control or USP19 silenced MDAMB231 cells at each wound-edge. (n=6, one-way ANOVA, Dunnett’s multiple comparison test. shRNA#1 p= 0.0003 and shRNA#2 p= 0.0527). ( F ) Rose diagrams representing the orientation of migration trajectories relative to cells position at time= 0 hours. Cells from the left wound-edge of three videos per cell line are shown, and each sector indicates the frequency of trajectory orientation. Control cell line (n= 131), shRNA#1 (n= 107) and shRNA#2 (n= 123). F: forward to direction of migration, L: left to direction of migration, O: opposite to direction of migration, R: right to direction of migration.

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: Generated, Expressing, Stable Transfection, Control, Plasmid Preparation, Transferring, Imaging, Wound Healing Assay, Comparison, shRNA, Migration

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: We generated USP10 silenced MDAMB231 cells and compared the effect on migration with USP19 depletion experiments. ( A ) Efficiencies of shRNA-mediated USP10 knockdown were confirmed by RT-PCR (n= 4, one-way ANOVA, Dunnett’s multiple comparison test. shRNA#1 p= 0.0004 and shRNA#2 p= 0.0002, relative to mRNA expression in control cells). ( B ) Wound healing assays were used to analyze migration. The graph shows the gap covered area for control or cells stably expression shRNAs targeting USP10 or USP19 after 8 hours (n≥4 Kruskal-Wallis, Dunn’s multiple comparison test. USP10 shRNA#1 p= 0.4576, USP10 shRNA#2 p= 0.0397, USP19 shRNA#1 p= 0.0141 and USP19 shRNA#2 p= 0.0060). ( C ) LRP6 protein expression was evaluated in control or USP10 silenced MDAMB231 cells. Left: representative image of a blot; right: LRP6 signal quantification (n=3, Kruskal-Wallis and Dunn’s multiple comparison test. shRNA#1 p> 0.9999 and shRNA#2 p> 0.9999).

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: Generated, Migration, shRNA, Knockdown, Reverse Transcription Polymerase Chain Reaction, Comparison, Expressing, Control, Stable Transfection

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: USP19 silencing effects were studied in MDAMB436 cells. ( A ) Left: Efficiencies of shRNA-mediated target gene knockdown were confirmed by RT-PCR (n≥ 5, Kruskal-Wallis, Dunn’s multiple comparison test. shRNA#1 p= 0.6028 and shRNA#2 p= 0.8335) and Right: Western Blotting (n= 4, one-way ANOVA, Dunnett’s multiple comparison test. shRNA#1 p= 0.0039 and shRNA#2 p= 0.0002). ( B ) Cells migratory potential was evaluated by wound healing assay. Left: gap covered area (mm 2 ) after 8 hours (n= 3, Kruskal-Wallis, Dunn’s multiple comparison test. shRNA#1 p= 0.0146 and shRNA#2 p= 0.3594); right: representative areas in a wound healing experiment at the indicated time points. Scale bar= 100 μm. ( C ) Invasiveness was assessed with Matrigel 3D experiments. Left: Colony area was calculated at the end of the experiment (n= 3, Kruskal-Wallis, Dunn’s multiple comparison test. shRNA#1 p= 0.2021 and shRNA#2 p= 0.0341); right: a representative area for the cell invasion assay after 5 days in culture is shown. Scale bar= 200 μm.

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: shRNA, Knockdown, Reverse Transcription Polymerase Chain Reaction, Comparison, Western Blot, Wound Healing Assay, Invasion Assay

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: ( A ) Different constructs of USP19 were overexpressed in MCF7 cells, and their expression confirmed by Western Blotting (n=4, Kruskal-Wallis, Dunn’s multiple comparison test. WT p>0.9999, C506S p= 0.0777 and WTΔTM p= 0.0070). ( B ) Migratory potential was evaluated by wound healing assay. Left: gap covered area (mm2) after 12 hours (n≥ 4, Kruskal-Wallis, Dunn’s multiple comparison test. WT p= 0.0107, C506S p>0.9999, WTΔTM p>0.9999) and right: representative areas in a wound healing experiment at the indicated time points. Scale bar= 100 μm. ( C ) Matrigel invasion was assessed over a 30 days period. Left: Representative brightfield images obtained at the end of the experiment using a 10X objective are shown; scale bar= 100 μm. Right: colony size was calculated (n≥ 4, Kruskal-Wallis, Dunn’s multiple comparison test. WT p= 0.0485, C506S p>0.9999, WTΔTM p>0.9999).

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: Construct, Expressing, Western Blot, Comparison, Wound Healing Assay

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: There are a variety of USP19 mRNA transcripts generated by alternative splicing, and not all of them show the same functional properties. In this regard, changes in the last exon coding sequence generate soluble isoforms and endoplasmic reticulum (ER) membrane-bound isoforms. In order to analyze whether USP19’s transmembrane domain was required for migration and 3D growth, we generated a GFP-tagged USP19 construct (wild type for its catalytic activity) with a point mutation that generates a premature stop codon. This mutation prevented the transmembrane domain to be translated, therefore generating a protein that mimicked USP19’s soluble isoform (named WTΔTM). We analyzed overexpressed-USP19 proteins subcellular localization by transiently transfecting U2OS cells with wild type (WT), catalytically dead mutant (C506S) or cytoplasmic (WTΔTM) GFP-tagged USP19 constructs. Cells were fixed with 4% paraformaldehyde, stained with DAPI and mounted. Images were captured using an inverted fluorescence microscopy. Scale bar= 100 μm.

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: Generated, Alternative Splicing, Functional Assay, Sequencing, Membrane, Migration, Construct, Activity Assay, Mutagenesis, Staining, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: ( A ) Downregulation of USP19 attenuates tumorigenicity in vivo: Control or USP19-silenced MDAMB231 cells were subcutaneously inoculated into the mammary fat pads of female NOD/SCID mice and tumor growth monitored every 2-3 days. Left: tumor volume was calculated at the indicated time points (results show mean value ± S.E.); right: Kaplan-Meier curves were built for Tumor Free Survival (TFS) over time (n≥ 10, Log-Rank (Mantel-Cox) test, shRNA#1 p< 0.0001 and shRNA#2 p< 0.0001). ( B ) Silencing effects of USP19 on experimental metastasis assays: NOD/SCID male mice were inoculated with MDAMB231 USP19-silenced cells through tail vein injection and after 2 months, lungs were harvested. Top left: metastatic foci were estimated by qPCR human DNA quantification (n≥ 6, Kruskal-Wallis, Dunn’s multiple comparison test. shRNA#1 p> 0.9999 and shRNA#2 p= 0.0032). Bottom left: representative lung images stained with Indian ink at the end of the experiment are shown. Scale bar= 1 cm. Right: metastatic load quantification was performed by evaluating lung Hematoxylin & Eosin stained slides. We used a lesion-based analysis of percent of metastatic pixels to compare the differences in metastatic load produced on the lungs by the MDAMB231 cell lines (n= 6, Kruskal-Wallis, Dunn’s multiple comparison test. shRNA#1 p> 0.9999 and shRNA#2 p= 0.0299). ( C ) USP19 catalytic activity is needed for tumorigenicity in vivo: control, WT or C506S mutant versions of USP19 overexpressing MCF7 cells were subcutaneously inoculated into the mammary fat pads of female NOD/SCID mice. Left: tumor volume was calculated at the indicated time points (results show mean value ± S.E.); right: Kaplan-Meier curves were built for Tumor Free Survival (TFS) over time (n≥ 7, Log-Rank (Mantel-Cox) test, WT p< 0.0001 and C506S p= 0.5307).

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: In Vivo, Control, shRNA, Injection, Comparison, Staining, Produced, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: ( A ) Downregulation of USP19 attenuates tumorigenicity in vivo: control or USP19-silenced MDAMB436 cells were subcutaneously inoculated into the mammary fat pads of female NOD/SCID mice. Top left: Tumor volume was measured at the indicated time points (results show mean value ± S.E.). Top right: the area under the tumor volume curves was calculated at the end of the experiment (n≥ 6, Kruskal-Wallis, Dunn’s multiple comparison test. shRNA#1 p= 0.0053 and shRNA#2 p= 0.0014). Bottom: Kaplan-Meier curves were built for Tumor Free Survival (TFS) over time (n≥ 6, Log-Rank (Mantel-Cox) test, shRNA#1 p= 0.0003 and shRNA#2 p= 0.0001). ( B ) Experimental metastasis assay: NOD/SCID male mice were inoculated with MDAMB436 USP19-silenced cells through tail vein injection and after 2 months, lungs were harvested. Metastasis foci were estimated by qPCR human DNA quantification (n≥ 2).

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: In Vivo, Control, Comparison, shRNA, Injection

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: Representative images of lung sections of mice injected with control or USP19-silenced MDAMB231 cells. Tissues were analyzed for metastatic load by Hematoxylin & Eosin staining ( A , C , and E ), and nodules tumor origin was confirmed by staining with the mouse anti-human CD44s monoclonal antibody ( B , D , and F ). Scale bar: 50 μm.

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: Injection, Control, Staining

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: Crystal violet (CV) staining was used to determine cell growth over time. Cells were seeded onto wells and allowed to attach. At the indicated time points, cells were fixed and then stained at the end of the experiment. ( A ) The graph shows the mean relative CV absorbance every 24 hours. ( B ) Doubling time was calculated for MCF7 cells overexpressing WT or mutant C506S USP19 constructs (n≥ 3, Mann-Whitney test, p= 0.4000).

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: Staining, Mutagenesis, Construct, MANN-WHITNEY

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: An in silico study was performed in order to analyze the relationship between USP19 expression levels and different pathway activation status. ( A ) USP19 mRNA expression among primary breast carcinomas according to their intrinsic subtype. Expression analysis showed a consistent up-regulation in luminal A and B subtypes compared with basal-like and Her2 subtypes. ( B ) Luminal A/B primary breast cancers divided into low (n= 77) or high (n= 209) USP19 mRNA expression levels. ( C ) Significantly activated pathways among Luminal A/B tumors with high USP19 mRNA expression (n≥ 77, SAM test, p< 0.01). Western blotting was performed in order to analyze LRP6 protein expression in breast cancer cells upon USP19 genetic manipulation. ( D ) Top: Western Blot quantification in control or USP19 silenced MDAMB231 cells (n=5, one-way ANOVA, Dunnett’s multiple comparison test. shRNA#1 p= 0.0492 and shRNA#2 p= 0.0226), bottom: representative image of a blot. ( E ) Top: Western Blot quantification in MCF7 cells overexpressing control or GFP-tagged USP19 constructs (n=5, one-way ANOVA, Dunnett’s multiple comparison test. WT p= 0.0484, C506S p= 0.8469 and WTΔTM p= 0.9968), bottom: representative image of a blot. ( F ) Wound healing assays were performed in order to analyze endogenous LRP6 silencing effects in MCF7 cells overexpressing WT or C506S mutant versions of USP19. Cells were stably transduced with control vector (‘ctrol’, PLKO.1 empty vector), or shRNAs targeting LRP6 (sh#1 and sh#2). Scratching with a pipette tip made a gap on a monolayer of the different cell cultures, and time-lapse imaging monitored the number of migrating cells across the border. The graph shows the gap covered area (mm 2 ) after 8 hours (n=3, Kruskal-Wallis and Dunn’s multiple comparison test for WT or C506S overexpressing MCF7 cell lines, analyzed separately. WT overexpressing MCF7 cell line: sh#1 p= 0.0341 and sh#2 p= 0.2021. C506S overexpressing MCF7 cell line: sh#1 p= 0.7422 and sh#2 p> 0.9999).

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: In Silico, Expressing, Activation Assay, Western Blot, Control, Comparison, shRNA, Construct, Mutagenesis, Stable Transfection, Transduction, Plasmid Preparation, Transferring, Imaging

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: ( A ) Western blotting was performed in order to analyze LRP6 protein expression in the breast cancer cell lines used in this manuscript. ( B ) LRP6 protein expression was evaluated in control or USP19 silenced MDAMB436 cells. Top: LRP6 signal quantification (n=5, one-way ANOVA, Dunnett’s multiple comparison test. shRNA#1 p= 0.0139 and shRNA#2 p= 0.0188), bottom: representative image of a blot.

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: Western Blot, Expressing, Control, Comparison, shRNA

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: Kaplan-Meier estimates of DFS and DRFS in patients with early breast cancer tumors, according to high (solid green lines) and low (dashed blue lines) expression of USP19. ( A ) 19 out of 62 patients (30.6%) harboring USP19 High tumors and 30 out of 106 patients (28.3%) with USP19 Low tumors had a disease relapse (n= 168, Log-Rank (Mantel-Cox) test, p= 0.817). ( B ) Distant metastases developed in 18 out of 62 (29.0%) and 11 out of 106 (10.4%) of patients with USP19 High and USP19 Low tumors, respectively (n= 168, Log-Rank (Mantel-Cox) test, p= 0.003).

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: Expressing

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: USP19 mRNA expression levels were analyzed by RT-qPCR in cultured MDAMB231 cells expressing PLKO.1 empty vector (growing in culture dishes), or in tumors that generated in NOD/SCID mice upon injection of MDAMB231 cells expressing PLKO.1 empty vector (n≥ 5, Mann-Whitney test, p= 0.0326).

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Plasmid Preparation, Generated, Injection, MANN-WHITNEY

Journal: bioRxiv

Article Title: USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

doi: 10.1101/2020.07.01.181883

Figure Lengend Snippet: Immunohistochemical staining: examples of ( A ) low and ( B ) high expression of USP19 in breast cancer. Scale bar: 200 μm.

Article Snippet: Membranes were incubated with primary antibodies: rabbit anti-USP19 antibody (Bethyl Cat# A301-587A, 1:2000 dilution), mouse anti-tubulin antibody (Santa Cruz Biotechnology, Cat# sc-398103, 1:3000 dilution), mouse anti-β-actin antibody (Cell Signaling, Cat# 3700, 1:5000 dilution), mouse anti-GFP antibody (Santa Cruz Biotechnology, Cat# sc-9996 B2, 1:1000 dilution) and rabbit anti-LRP6 antibody (Cell Signaling Cat#2560 C5C7, 1:2000 dilution) and HRP-conjugated secondary antibodies: anti-rabbit (GE Healthcare Cat# NA934); anti-mouse (GE Healthcare Cat# NA931, 1:5000 dilution), and then detected using an ECL SuperSignal West Femto y West Pico detection kit (Pierce).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Univariate Cox Model for the Association Between Survival and Clinicopathological Features in GC

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Relative expression of USP19 in GC cell lines and GES1. ( A ) Differential expression of USP19 mRNA in gastric cell lines by real-time PCR assay. ( B ) The differential expression of USP19 protein in gastric cell lines by WB analysis. ( C ) Differential expression and distribution of USP19 protein in gastric cell lines by confocal analysis, Scale bar=20 μm. ( D ) Relative expression of USP19 in paired 212 GC samples and ANTs by IHC staining and analysis by Chi-square test. ( E ) USP19 protein levels in GC tissues and ANTs were analyzed by IHC staining (200× magnification). High or low expression of USP19 protein in ANTs (a and b); high or low expression of USP19 protein in intestinal-type GC (c and d); high or low expression of USP19 protein in diffuse-type GC (e and f), Scale bar=200 μm.

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

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

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: The Relationship Between USP19 Expression and Other Clinicopathological Parameters in GC

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Kaplan–Meier survival curves of GC patients according to the clinical features and expression of USP19. ( A ) Patients with high-level USP19 expression showed poor survival. ( B ) TNM stage was a promising indicator of the prognosis of GC patients. ( C ) Patients with intestinal-type GC had better survival than those with diffuse-type GC. ( D ) Low-level USP19 expression predicted better survival in patients with TNM stage III. ( E ) Low-level USP19 expression predicted better survival in patients with diffuse-type GC. ( F ) Low-level USP19 expression predicted better survival in patients with intestinal-type GC.

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Multivariate Cox Model for the Association Between Survival and Clinicopathological Factors in GC

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Functional roles of USP19 in cell models. ( A ) Knockdown of USP19 inhibited SGC7901 cell growth, as determined by MTT assay. Bars: SD; **P<0.01. ( B ) Knockdown of USP19 decreased the level of MMP2 and MMP9 and increased the level of the apoptotic-related protein, cleaved caspase-3, in SGC7901 cells. ( C ) Knockdown of USP19 inhibited colony formation in SGC7901 cells. The data are shown as Mean±SD (n=3). **P<0.01. ( D ) Ectopic expression of USP19 promoted GES1 cell growth, as shown by MTT assay. Bars: SD; **P<0.01. ( E ) Overexpression of USP19 increased the protein level of MMP2 and MMP9 and decreased the level of the apoptotic-related protein, cleaved caspase-3. ( F ) Ectopic expression of USP19 increased colony formation in GES1 cells. The data are shown as Mean±SD (n=3). **P<0.01. All WB experiments were repeated three times.

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Functional Assay, MTT Assay, Expressing, Over Expression

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Effect of USP19 on cell invasion and migration and MMP2/MMP9 enzyme activity. ( A ) Knockdown of USP19 reduced SGC7901 cell invasion. Images were acquired at the indicated time (200× magnification). Data are shown as Mean±SD (n=5). **P<0.01. ( B ) Knockdown of USP19 reduced the migration of SGC7901 cells, as determined by wound healing assay at 0, 12, 24, and 36 h. Images were acquired at the indicated time (100× magnification). Data are shown as Mean±SD (n=3). **P<0.01. ( C ) SGC7901 cells were transfected with ShUSP19 or vector for 36 h and then subjected to gelatin zymography to analyze the activity of MMP2 and MMP9. ( D ) Ectopic expression of USP19 enhanced invasion of GES1 cells. Images were acquired at the indicated time (200× magnification). Data are shown as Mean±SD (n=5). **P<0.01. ( E ) Ectopic expression of USP19 increased the migration of GES1 cells, as determined by wound healing assay at 0, 12, 24, and 36 h. Images were acquired at the indicated time (100× magnification). Data are shown as Mean±SD (n=3). **P<0.01. ( F ) GES1 cells were transfected with USP19-overexpressing plasmid or control vector for 36 h and then subjected to gelatin zymography to analyze the activity of MMP2 and MMP9.

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Migration, Activity Assay, Wound Healing Assay, Transfection, Plasmid Preparation, Zymography, Expressing

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Knockdown of USP19 reduced gastric carcinogenesis in animal models. Knockdown of USP19 inhibited tumor growth. Five nude mice were injected subcutaneously with 1 × 10 7 cells/mouse for each of the indicated stable cell lines of SGC7901-ShUSP19 or SGC7901-Vector, respectively. Results were presented as tumor volume at the different time points ( C ) and isolated xenografts at day 24 ( A ) with no change in body weight for each group ( B ). **P<0.01. ( D ) Tumors were isolated, fixed, and subjected to IHC assay (400× magnification).

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Injection, Stable Transfection, Plasmid Preparation, Isolation

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Prognostic roles of USP19 was determined in www.kmplot.com and correlation between mRNA levels of USP19 and MMP2/MMP9 in GC dataset from GEPIA Website ( http://gepia2.cancer-pku.cn ). Overall survival curves were plotted for all GC patients (n=876) with ( A ) different levels of USP19 expression; ( B ) Lauren’s classification; ( C and D ) HER2 status, ( E ) Different treatments, 5-FU based chemical therapy, ( F ) Surgery alone. From these data, low level of USP19 expression was correlated with better OS. The mRNA level of USP19 expression was positive association with MMP2/MMP9 expression. ( G) P<0.001 correlation between MMP9 and USP19; ( H ) P=0.062, correlation between MMP2 and USP19. Especially, the moderate correlation was performed between USP19 and MMP9 expression (Spearman’s R=0.24).

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Univariate Cox Model for the Association Between Survival and Clinicopathological Features in GC

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Relative expression of USP19 in GC cell lines and GES1. ( A ) Differential expression of USP19 mRNA in gastric cell lines by real-time PCR assay. ( B ) The differential expression of USP19 protein in gastric cell lines by WB analysis. ( C ) Differential expression and distribution of USP19 protein in gastric cell lines by confocal analysis, Scale bar=20 μm. ( D ) Relative expression of USP19 in paired 212 GC samples and ANTs by IHC staining and analysis by Chi-square test. ( E ) USP19 protein levels in GC tissues and ANTs were analyzed by IHC staining (200× magnification). High or low expression of USP19 protein in ANTs (a and b); high or low expression of USP19 protein in intestinal-type GC (c and d); high or low expression of USP19 protein in diffuse-type GC (e and f), Scale bar=200 μm.

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Expressing, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Immunohistochemistry

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: The Relationship Between USP19 Expression and Other Clinicopathological Parameters in GC

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Kaplan–Meier survival curves of GC patients according to the clinical features and expression of USP19. ( A ) Patients with high-level USP19 expression showed poor survival. ( B ) TNM stage was a promising indicator of the prognosis of GC patients. ( C ) Patients with intestinal-type GC had better survival than those with diffuse-type GC. ( D ) Low-level USP19 expression predicted better survival in patients with TNM stage III. ( E ) Low-level USP19 expression predicted better survival in patients with diffuse-type GC. ( F ) Low-level USP19 expression predicted better survival in patients with intestinal-type GC.

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Multivariate Cox Model for the Association Between Survival and Clinicopathological Factors in GC

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Functional roles of USP19 in cell models. ( A ) Knockdown of USP19 inhibited SGC7901 cell growth, as determined by MTT assay. Bars: SD; **P<0.01. ( B ) Knockdown of USP19 decreased the level of MMP2 and MMP9 and increased the level of the apoptotic-related protein, cleaved caspase-3, in SGC7901 cells. ( C ) Knockdown of USP19 inhibited colony formation in SGC7901 cells. The data are shown as Mean±SD (n=3). **P<0.01. ( D ) Ectopic expression of USP19 promoted GES1 cell growth, as shown by MTT assay. Bars: SD; **P<0.01. ( E ) Overexpression of USP19 increased the protein level of MMP2 and MMP9 and decreased the level of the apoptotic-related protein, cleaved caspase-3. ( F ) Ectopic expression of USP19 increased colony formation in GES1 cells. The data are shown as Mean±SD (n=3). **P<0.01. All WB experiments were repeated three times.

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Functional Assay, Knockdown, MTT Assay, Expressing, Over Expression

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Effect of USP19 on cell invasion and migration and MMP2/MMP9 enzyme activity. ( A ) Knockdown of USP19 reduced SGC7901 cell invasion. Images were acquired at the indicated time (200× magnification). Data are shown as Mean±SD (n=5). **P<0.01. ( B ) Knockdown of USP19 reduced the migration of SGC7901 cells, as determined by wound healing assay at 0, 12, 24, and 36 h. Images were acquired at the indicated time (100× magnification). Data are shown as Mean±SD (n=3). **P<0.01. ( C ) SGC7901 cells were transfected with ShUSP19 or vector for 36 h and then subjected to gelatin zymography to analyze the activity of MMP2 and MMP9. ( D ) Ectopic expression of USP19 enhanced invasion of GES1 cells. Images were acquired at the indicated time (200× magnification). Data are shown as Mean±SD (n=5). **P<0.01. ( E ) Ectopic expression of USP19 increased the migration of GES1 cells, as determined by wound healing assay at 0, 12, 24, and 36 h. Images were acquired at the indicated time (100× magnification). Data are shown as Mean±SD (n=3). **P<0.01. ( F ) GES1 cells were transfected with USP19-overexpressing plasmid or control vector for 36 h and then subjected to gelatin zymography to analyze the activity of MMP2 and MMP9.

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Migration, Activity Assay, Knockdown, Wound Healing Assay, Transfection, Plasmid Preparation, Zymography, Expressing, Control

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Knockdown of USP19 reduced gastric carcinogenesis in animal models. Knockdown of USP19 inhibited tumor growth. Five nude mice were injected subcutaneously with 1 × 10 7 cells/mouse for each of the indicated stable cell lines of SGC7901-ShUSP19 or SGC7901-Vector, respectively. Results were presented as tumor volume at the different time points ( C ) and isolated xenografts at day 24 ( A ) with no change in body weight for each group ( B ). **P<0.01. ( D ) Tumors were isolated, fixed, and subjected to IHC assay (400× magnification).

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Knockdown, Injection, Stable Transfection, Plasmid Preparation, Isolation

Journal: OncoTargets and therapy

Article Title: USP19 Enhances MMP2/MMP9-Mediated Tumorigenesis in Gastric Cancer

doi: 10.2147/OTT.S240543

Figure Lengend Snippet: Prognostic roles of USP19 was determined in www.kmplot.com and correlation between mRNA levels of USP19 and MMP2/MMP9 in GC dataset from GEPIA Website ( http://gepia2.cancer-pku.cn ). Overall survival curves were plotted for all GC patients (n=876) with ( A ) different levels of USP19 expression; ( B ) Lauren’s classification; ( C and D ) HER2 status, ( E ) Different treatments, 5-FU based chemical therapy, ( F ) Surgery alone. From these data, low level of USP19 expression was correlated with better OS. The mRNA level of USP19 expression was positive association with MMP2/MMP9 expression. ( G) P<0.001 correlation between MMP9 and USP19; ( H ) P=0.062, correlation between MMP2 and USP19. Especially, the moderate correlation was performed between USP19 and MMP9 expression (Spearman’s R=0.24).

Article Snippet: The specific ectopic expression USP19 plasmid (RC214190, USP19 [Myc-DDK-tagged]-Human USP19, NM_006677, transcript variant 4) with the vector control (pCMV6-Entry, CAT#: PS10,0001) were purchased from a commercial company (OriGene Ltd., Rockville, MD), and these plasmids were transfected into GES1 cells with 400μg/mL neomycin (Gibco) addition to form stable clones.

Techniques: Expressing