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Addgene inc mammalian expression constructs prk flag usp19 wild type
Mammalian Expression Constructs Prk Flag Usp19 Wild Type, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Characterization of Ubiquitin Carboxyl-Terminal Hydrolase 19 Deficient Cells Reveals a Role for USP19 in the Secretion of Lysosomal Proteins

doi: 10.1016/j.mcpro.2024.100854

Figure Lengend Snippet: List of antibodies used in this study

Article Snippet: Two different constructs were used to rescue USP19 expression in USP19KO HEK cells, pRK-FLAG-USP19 (plasmid # 78597 from Addgene), and pEGFP-rUSP19ER (plasmid # 155247, Addgene), while pCMV3-C-GFPSpark (SinoBiological Eschborn) was used as a control.

Techniques: Ubiquitin Proteomics

A high-resolution mass spectrometry–based approach identifies proteins differentially abundant in the secretome of USP19 KO HEK cells. A , volcano plot showing the −Log10 of p -values versus the Log2 of protein ratio between USP19KO HEK (USP19KO) and USP19WT HEK cells of 1676 proteins (n = 6). Proteins significantly more abundant in the secretome of USP19KO cells are displayed as red filled dots, while less abundant proteins are displayed as blue filled dots. The solid black hyperbolic curves display the FDR at 0.05 and the dashed curves at 0.01. Unaltered proteins are displayed as the gray dots below the FDR curve. The Venn diagram in the inset displays the number of proteins identified in the secretome of WT or USP19KO cells and the number of proteins found in both secretomes. B , Western blot showing levels of USP19 in WT and USP19KO HEKs (Please note that this blot was made using the anti-USP19 clone A301-586A from Bethyl Laboratories, which was eventually discontinued; all other USP19 blots shown in this study were obtained with the anti-USP19 clone A301-587A from the same vendor). C , number and subcellular location of proteins detected in the secretome of USP19KO cells. D , gene ontology and ( E ) KEGG pathways analysis of proteins regulated (FDR p = 0.05) in USP19KO cells to identify biological functions of USP19.

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Characterization of Ubiquitin Carboxyl-Terminal Hydrolase 19 Deficient Cells Reveals a Role for USP19 in the Secretion of Lysosomal Proteins

doi: 10.1016/j.mcpro.2024.100854

Figure Lengend Snippet: A high-resolution mass spectrometry–based approach identifies proteins differentially abundant in the secretome of USP19 KO HEK cells. A , volcano plot showing the −Log10 of p -values versus the Log2 of protein ratio between USP19KO HEK (USP19KO) and USP19WT HEK cells of 1676 proteins (n = 6). Proteins significantly more abundant in the secretome of USP19KO cells are displayed as red filled dots, while less abundant proteins are displayed as blue filled dots. The solid black hyperbolic curves display the FDR at 0.05 and the dashed curves at 0.01. Unaltered proteins are displayed as the gray dots below the FDR curve. The Venn diagram in the inset displays the number of proteins identified in the secretome of WT or USP19KO cells and the number of proteins found in both secretomes. B , Western blot showing levels of USP19 in WT and USP19KO HEKs (Please note that this blot was made using the anti-USP19 clone A301-586A from Bethyl Laboratories, which was eventually discontinued; all other USP19 blots shown in this study were obtained with the anti-USP19 clone A301-587A from the same vendor). C , number and subcellular location of proteins detected in the secretome of USP19KO cells. D , gene ontology and ( E ) KEGG pathways analysis of proteins regulated (FDR p = 0.05) in USP19KO cells to identify biological functions of USP19.

Techniques: Mass Spectrometry, Western Blot

Subcellular location analysis of proteins secreted by USP19KO HEK cells . A , analysis of subcellular locations among proteins regulated in USP19KO cells. For each displayed subcellular location, the ratio between the number of regulated proteins with a specific subcellular location (RegSL) and the total number of regulated proteins (RegTot) was normalized in a logarithmic scale against the ratio between the total number of proteins with a specific subcellular location (TOTSL) and the total number of proteins detected in the secretome (TOT). B , analysis of changes in subcellular locations among proteins regulated in USP19KO cells. For each displayed subcellular location, the ratio between the number of upregulated and downregulated proteins with a specific subcellular location (Up SL and Down SL, respectively) was normalized in a logarithmic scale against the ratio between the total number of upregulated proteins (UP tot) and downregulated proteins (Down tot). Nanoparticle Tracking Analysis of the number ( C ) and size ( D ) of extracellular vesicles released by USP19WT or KO HEK293T cells. Western blots ( E ) and relative band quantifications ( F ) of LAMP1, p62, and LC3B in the lysate of WT and USP19KO HEKs. Loss of USP19 in KO cells is also displayed (∗ indicates a nonspecific band that does not disappear when USP19 is ablated), and actin was used as a loading control.

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Characterization of Ubiquitin Carboxyl-Terminal Hydrolase 19 Deficient Cells Reveals a Role for USP19 in the Secretion of Lysosomal Proteins

doi: 10.1016/j.mcpro.2024.100854

Figure Lengend Snippet: Subcellular location analysis of proteins secreted by USP19KO HEK cells . A , analysis of subcellular locations among proteins regulated in USP19KO cells. For each displayed subcellular location, the ratio between the number of regulated proteins with a specific subcellular location (RegSL) and the total number of regulated proteins (RegTot) was normalized in a logarithmic scale against the ratio between the total number of proteins with a specific subcellular location (TOTSL) and the total number of proteins detected in the secretome (TOT). B , analysis of changes in subcellular locations among proteins regulated in USP19KO cells. For each displayed subcellular location, the ratio between the number of upregulated and downregulated proteins with a specific subcellular location (Up SL and Down SL, respectively) was normalized in a logarithmic scale against the ratio between the total number of upregulated proteins (UP tot) and downregulated proteins (Down tot). Nanoparticle Tracking Analysis of the number ( C ) and size ( D ) of extracellular vesicles released by USP19WT or KO HEK293T cells. Western blots ( E ) and relative band quantifications ( F ) of LAMP1, p62, and LC3B in the lysate of WT and USP19KO HEKs. Loss of USP19 in KO cells is also displayed (∗ indicates a nonspecific band that does not disappear when USP19 is ablated), and actin was used as a loading control.

Techniques: Western Blot, Control

Quantitative proteomics identifies proteins secreted in a USP19-dependent manner. A , Venn diagram showing the number of proteins identified by mass spectrometry analysis in the conditioned media and/or lysates of USP19KO cells. B , graphic representation of protein changes of the 1274 proteins identified in both conditioned media and lysates of USP19KO cells. Proteins have been divided in nine subgroups based on their increase or decrease in the conditioned media and lysates of USP19KO cells. Proteins decreased in the conditioned media and not reduced in the cell lysate (proteins potentially secreted in a USP19-dependent manner) are listed, with lysosome-resident proteins written in bold (according to UniProt annotation - GO 0005764)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Characterization of Ubiquitin Carboxyl-Terminal Hydrolase 19 Deficient Cells Reveals a Role for USP19 in the Secretion of Lysosomal Proteins

doi: 10.1016/j.mcpro.2024.100854

Figure Lengend Snippet: Quantitative proteomics identifies proteins secreted in a USP19-dependent manner. A , Venn diagram showing the number of proteins identified by mass spectrometry analysis in the conditioned media and/or lysates of USP19KO cells. B , graphic representation of protein changes of the 1274 proteins identified in both conditioned media and lysates of USP19KO cells. Proteins have been divided in nine subgroups based on their increase or decrease in the conditioned media and lysates of USP19KO cells. Proteins decreased in the conditioned media and not reduced in the cell lysate (proteins potentially secreted in a USP19-dependent manner) are listed, with lysosome-resident proteins written in bold (according to UniProt annotation - GO 0005764)

Techniques: Quantitative Proteomics, Mass Spectrometry

USP19 regulates the secretion of LGMN in different cell types. A and B , immunoblots showing protein abundance ( A ) and relative densitometric quantifications ( B ) of LGMN in the conditioned media and lysates of USP19KO HEK cells, transfected either with GFP-USP19, GFP, or FLAG-USP19. Expression of USP19 in the KO cells increased levels of LGMN in the conditioned media compared to GFP expressing control cells, indicating that ectopic expression of USP19 rescued secretion of the two proteins (densitometric quantifications shown as mean values ± SD; ∗∗ represents p < 0.01, Student’s t test; three independent experiments are displayed out of six quantified, n = 6). Clusterin was used as an example of protein that is secreted into the conditioned media independently from USP19. Levels of USP19 in the lysate of transfected cells are displayed, together with actin that was used as a loading control. C – F , immunoblots and their relative band densitometric quantifications showing LGMN in USP19 knockdown or control HTB94 ( C ), HeLa ( D ) U251 ( E ), and LX-2 cells ( F ). For each cell type, levels of USP19 in the lysate were shown. Actin was used as a loading control. USP19 silencing in these cells led to a decrease of LGMN in the conditioned media, while it did not alter its cellular levels (densitometric quantifications shown as mean values ± SD; ∗ represents p < 0.05; ∗∗ represents p < 0.01; and ∗∗∗ represents p < 0.005, Student’s t test; three independent experiments are displayed out of six quantified, n = 6).

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Characterization of Ubiquitin Carboxyl-Terminal Hydrolase 19 Deficient Cells Reveals a Role for USP19 in the Secretion of Lysosomal Proteins

doi: 10.1016/j.mcpro.2024.100854

Figure Lengend Snippet: USP19 regulates the secretion of LGMN in different cell types. A and B , immunoblots showing protein abundance ( A ) and relative densitometric quantifications ( B ) of LGMN in the conditioned media and lysates of USP19KO HEK cells, transfected either with GFP-USP19, GFP, or FLAG-USP19. Expression of USP19 in the KO cells increased levels of LGMN in the conditioned media compared to GFP expressing control cells, indicating that ectopic expression of USP19 rescued secretion of the two proteins (densitometric quantifications shown as mean values ± SD; ∗∗ represents p < 0.01, Student’s t test; three independent experiments are displayed out of six quantified, n = 6). Clusterin was used as an example of protein that is secreted into the conditioned media independently from USP19. Levels of USP19 in the lysate of transfected cells are displayed, together with actin that was used as a loading control. C – F , immunoblots and their relative band densitometric quantifications showing LGMN in USP19 knockdown or control HTB94 ( C ), HeLa ( D ) U251 ( E ), and LX-2 cells ( F ). For each cell type, levels of USP19 in the lysate were shown. Actin was used as a loading control. USP19 silencing in these cells led to a decrease of LGMN in the conditioned media, while it did not alter its cellular levels (densitometric quantifications shown as mean values ± SD; ∗ represents p < 0.05; ∗∗ represents p < 0.01; and ∗∗∗ represents p < 0.005, Student’s t test; three independent experiments are displayed out of six quantified, n = 6).

Techniques: Western Blot, Quantitative Proteomics, Transfection, Expressing, Control, Knockdown

LGMN is secreted through an USP19-dependent mechanism that requires PI3K and the fusion of intracellular pH sensitive vesicles with the plasma membrane . Immunoblots showing protein abundance and relative densitometric quantifications of LGMN in the conditioned media and cell lysates of WT and USP19KO HEK cells, treated with or without 350 nM brefeldin A (BFA) ( A and B ), 10 mM 3-methyladenosine (3-MA) ( C and D ) or 10 nM chloroquine (CQ) ( E and F ), 10 μM GW4869 ( G and H ), and 80 μM dynasore for 24 h (I-J). Clusterin (CLU), a protein known to be secreted in a conventional manner, was used to show effectiveness of the BFA treatment and actin was used as a loading control in ( A ). LC3-I to LC3-II conversion was observed in 3-MA–treated cells to confirm reduction in autophagosome formation in the presence of the inhibitor, and actin was used as a loading control in ( C ). LC3-I to LC3-II conversion was analysed to confirm inhibition of lysosomal activity in the presence of CQ, and actin was used as a loading control in ( E ).

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Characterization of Ubiquitin Carboxyl-Terminal Hydrolase 19 Deficient Cells Reveals a Role for USP19 in the Secretion of Lysosomal Proteins

doi: 10.1016/j.mcpro.2024.100854

Figure Lengend Snippet: LGMN is secreted through an USP19-dependent mechanism that requires PI3K and the fusion of intracellular pH sensitive vesicles with the plasma membrane . Immunoblots showing protein abundance and relative densitometric quantifications of LGMN in the conditioned media and cell lysates of WT and USP19KO HEK cells, treated with or without 350 nM brefeldin A (BFA) ( A and B ), 10 mM 3-methyladenosine (3-MA) ( C and D ) or 10 nM chloroquine (CQ) ( E and F ), 10 μM GW4869 ( G and H ), and 80 μM dynasore for 24 h (I-J). Clusterin (CLU), a protein known to be secreted in a conventional manner, was used to show effectiveness of the BFA treatment and actin was used as a loading control in ( A ). LC3-I to LC3-II conversion was observed in 3-MA–treated cells to confirm reduction in autophagosome formation in the presence of the inhibitor, and actin was used as a loading control in ( C ). LC3-I to LC3-II conversion was analysed to confirm inhibition of lysosomal activity in the presence of CQ, and actin was used as a loading control in ( E ).

Techniques: Clinical Proteomics, Membrane, Western Blot, Quantitative Proteomics, Control, Inhibition, Activity Assay

Secretion of lysosomal proteins is not regulated by their deubiquitination by USP19 or direct interaction with the DUB. A , cell lysates and immunoprecipitates from HEK cells transfected with USP19-FLAG, MXRA8-FLAG, or an empty vector were immunoblotted for LGMN and the known USP19 interactor HSP90. β-actin was used as a loading control. B , volcano plot showing the −Log10 of p -values versus the Log2 of protein ratio of 5208 proteins between the immunoprecipitates from USP19-FLAG– and FLAG-MXRA8–transfected HEK cells. USP19 and MXRA8 are displayed as filled red dots. Significantly altered proteins ( i.e. proteins above the FDR curves) are displayed as filled black dots , and those with a Log2 USP19/MXRA8 ratio above 3 (putative USP19 interactors) are displayed with blue dots . Not altered proteins are displayed as open gray circles. C , immunoblots showing LGMN in WT and USP19KO HEKs, in the presence or absence of the proteasome inhibitor MG132. Ubiquitin (Ub) was used as a control for MG132 treatment, and β-actin as a loading control. D and E , ubiquitinated proteins enriched from WT and USP19KO cells, treated with or without MG132, were analyzed by immunoblotting and unbiased proteomics. D , immunoblots showing ubiquitinated LGMN and p62 in WT and USP19KO HEKs, either in the presence or absence of MG132. E , volcano plots showing the −Log10 of p -values versus the Log2 of protein ratio between USP19KO and WT cells, treated with MG132 to inhibit proteasomal degradation, of 460 proteins (n = 3). Glypican 6 (GPC6) was the only significantly regulated protein and it is displayed as a red filled dot above the FDR ( black hyperbolic curves ). Not altered proteins are displayed as gray dots below the FDR. F , quantification of p62 bands from the immunoblot shown in ( D ) (n = 3; ∗ represents p < 0.05; Student’s t test). G and H , flow cytometry analysis shows that levels of LAMP1 on the cell surface of WT HEKs were higher than on USP19KO HEKs. Flow cytometry histograms are shown in ( G ), and quantification of three separate experiments in ( H ) (∗∗ represents p < 0.01, Student t test).

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Characterization of Ubiquitin Carboxyl-Terminal Hydrolase 19 Deficient Cells Reveals a Role for USP19 in the Secretion of Lysosomal Proteins

doi: 10.1016/j.mcpro.2024.100854

Figure Lengend Snippet: Secretion of lysosomal proteins is not regulated by their deubiquitination by USP19 or direct interaction with the DUB. A , cell lysates and immunoprecipitates from HEK cells transfected with USP19-FLAG, MXRA8-FLAG, or an empty vector were immunoblotted for LGMN and the known USP19 interactor HSP90. β-actin was used as a loading control. B , volcano plot showing the −Log10 of p -values versus the Log2 of protein ratio of 5208 proteins between the immunoprecipitates from USP19-FLAG– and FLAG-MXRA8–transfected HEK cells. USP19 and MXRA8 are displayed as filled red dots. Significantly altered proteins ( i.e. proteins above the FDR curves) are displayed as filled black dots , and those with a Log2 USP19/MXRA8 ratio above 3 (putative USP19 interactors) are displayed with blue dots . Not altered proteins are displayed as open gray circles. C , immunoblots showing LGMN in WT and USP19KO HEKs, in the presence or absence of the proteasome inhibitor MG132. Ubiquitin (Ub) was used as a control for MG132 treatment, and β-actin as a loading control. D and E , ubiquitinated proteins enriched from WT and USP19KO cells, treated with or without MG132, were analyzed by immunoblotting and unbiased proteomics. D , immunoblots showing ubiquitinated LGMN and p62 in WT and USP19KO HEKs, either in the presence or absence of MG132. E , volcano plots showing the −Log10 of p -values versus the Log2 of protein ratio between USP19KO and WT cells, treated with MG132 to inhibit proteasomal degradation, of 460 proteins (n = 3). Glypican 6 (GPC6) was the only significantly regulated protein and it is displayed as a red filled dot above the FDR ( black hyperbolic curves ). Not altered proteins are displayed as gray dots below the FDR. F , quantification of p62 bands from the immunoblot shown in ( D ) (n = 3; ∗ represents p < 0.05; Student’s t test). G and H , flow cytometry analysis shows that levels of LAMP1 on the cell surface of WT HEKs were higher than on USP19KO HEKs. Flow cytometry histograms are shown in ( G ), and quantification of three separate experiments in ( H ) (∗∗ represents p < 0.01, Student t test).

Techniques: Transfection, Plasmid Preparation, Control, Western Blot, Ubiquitin Proteomics, Flow Cytometry

Univariate Cox Model for the Association Between Survival and Clinicopathological Features in GC

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

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.

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.

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

The Relationship Between USP19 Expression and Other Clinicopathological Parameters in GC

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

Techniques: Expressing

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.

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.

Techniques: Expressing

Multivariate Cox Model for the Association Between Survival and Clinicopathological Factors in GC

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

Techniques: Expressing

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.

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.

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

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.

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.

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

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).

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).

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

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).

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).

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