human dusp10 mkp5  (Addgene inc)

Journal: Cancers

Article Title: DUSP10 Is a Regulator of YAP1 Activity Promoting Cell Proliferation and Colorectal Cancer Progression

doi: 10.3390/cancers11111767

Figure Lengend Snippet: Dual-specificity phosphatase 10 (DUSP10) expression promotes higher colorectal cancer (CRC) cell proliferation and in vivo tumor growth. ( a ) Total cell number of HT29lucD6-DUSP10 was normalized to HT29lucD6-EV. Two-way ANOVA followed by Bonferroni’s post-test (mean ± standard error of mean (SEM); *** p < 0.001) and eight independent experiments were performed. ( b ) Growth curves of HT29lucD6-EV and HT29lucD6-DUSP10 for 42 h using real-time proliferation analysis by xCELLigence technology. Linear regression analysis was performed (*** p < 0.001). Representative graph of six independent experiments. ( c ) Total cell number of HT29lucD6-shDUSP10 cell lines was normalized to HT29lucD6-SCR. Two-way ANOVA followed by Bonferroni’s post-test (mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001) and seven independent experiments were performed. ( d ) Growth curves of HT29lucD6-shDUSP10 and HT29lucD6-SCR for 42 h using real-time proliferation analysis by xCELLigence technology. Linear regression analysis was performed (** p < 0.01, *** p < 0.001). Representative graph of three independent experiments. ( e ) Bioluminescence imaging (BLI) of mice xenoinjected with HT29lucD6-DUSP10 and HT29lucD6-EV. Data was normalized to first week post-inoculation for each cell line. Two-way ANOVA followed by Bonferroni’s multiple comparison and linear regression analysis were performed (mean ± SEM; p < 0.05; 7–8 mice per group). ( f ) Tumor volume of HT29lucD6-DUSP10 and HT29lucD6-EV xenografts was measured for seven weeks. Two-way ANOVA followed by Bonferroni’s multiple comparison tests were performed (mean ± SEM; p < 0.05; five mice per group). ( g ) BLI of mice xenoinjected with HT29lucD6-shDUSP10 and HT29lucD6-SCR. Two-way ANOVA with Bonferroni’s multiple comparison test and linear regression analysis were performed (mean ± SEM; *** p < 0.001; eight mice per group). ( h ) Tumor volume of HT29lucD6-shDUSP10 and HT29lucD6-SCR xenografts was measured for seven weeks. Two-way ANOVA and Bonferroni’s multiple comparison test were performed (mean ± SEM; *** p < 0.001; four mice per group).

Article Snippet: Human DUSP10 cDNA (SC31766, OriGene Technologies, Rockville, MD, USA) was subcloned in the pLenti-CMV/TO-Hygro vector using specific primers. pLenti-KO.1-Puro carrying specific shRNA sequences were used to knockdown DUSP10 constructs (shDUSP10).

Techniques: Expressing, In Vivo, Imaging

Journal: Cancers

Article Title: DUSP10 Is a Regulator of YAP1 Activity Promoting Cell Proliferation and Colorectal Cancer Progression

doi: 10.3390/cancers11111767

Figure Lengend Snippet: Nuclear DUSP10 and Yes-associated protein 1 (YAP1) are increased in high-density conditions. ( a ) DUSP10 mRNA was quantified by HT29 in low density (LD) and high density (HD). Student’s t -test (mean ± SEM; *** p < 0.001) and four independent experiments were performed. ( b ) Expression of DUSP10, p-p38, and p38 of HT29 in LD and HD. (Left) A representative image of five independent experiments. (Right) Quantification of blots performed (mean ± SEM; Student’s t -test; ** p < 0.01, *** p < 0.001). ( c ) YAP1 mRNA was quantified from HT29 in LD and HD. Student’s t -test (mean ± SEM; * p < 0.05) and three independent experiments were performed. ( d ) Expression of YAP1 and p-YAP Ser127 of HT29 in LD and HD. (Left) A representative image of five independent experiments. (Right) Quantification of blots performed (mean ± SEM; Student’s t -test; ** p < 0.01, *** p < 0.001). ( e ) Expression of DUSP10, YAP1, and p-YAP Ser127 proteins from nuclear and cytoplasmic extracts from HT29lucD6-DUSP10 and HT29lucD6-EV in HD. LAMIN A/C and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) are nuclear and cytoplasm control proteins, respectively. (Left) A representative image of three independent experiments. (Right) Quantification of all blots performed (mean ± SEM; Student’s t -test; p < 0.05). ( f ) Expression of DUSP10, YAP1, and p-YAP Ser127 proteins from nuclear and cytoplasmic extracts of HT29lucD6-shDUSP10 and HT29lucD6-SCR in HD. LAMIN A/C and GAPDH/ACTIN are nuclear and cytoplasm control proteins, respectively. (Left) A representative image of three independent experiments. (Right) Quantification of all blots performed (mean ± SEM; Student’s t -test; * p < 0.05, ** p < 0.01, *** p < 0.001). Completed immunoblots of b,d,e,f are in , respectively.

Article Snippet: Human DUSP10 cDNA (SC31766, OriGene Technologies, Rockville, MD, USA) was subcloned in the pLenti-CMV/TO-Hygro vector using specific primers. pLenti-KO.1-Puro carrying specific shRNA sequences were used to knockdown DUSP10 constructs (shDUSP10).

Techniques: Expressing, Western Blot

Journal: Cancers

Article Title: DUSP10 Is a Regulator of YAP1 Activity Promoting Cell Proliferation and Colorectal Cancer Progression

doi: 10.3390/cancers11111767

Figure Lengend Snippet: A YAP inhibitor prevents DUSP10-enhanced proliferation. ( a ) Growth curves of HT29 proliferative response to SB239063 (SB 1 µM) and verteporfin (VP 1 µM) for 40 h after 2 h seeding (▼) using real-time proliferation analysis by xCELLigence technology. Linear regression analysis was performed (*** p < 0.001). Representative graph of four independent experiments. ( b ) Expression of DUSP10, YAP1, p-p38, and p38 protein levels in HT29 treated with SB (1 µM) and VP (1 µM) for 24 h (dotted line within a). (Left) A representative image of three independent experiments. (Right) Quantification of all blots performed (mean ± SEM; Student’s t -test; * p < 0.05, ** p < 0.01). ( c ) Growth curves of HT29-EV and HT29-DUSP10 proliferative response to SB 1 µM and DMSO for 40 h after 2 h seeding (▼) using real-time proliferation analysis by xCELLigence technology. Linear regression analysis was performed (*** p < 0.001). Representative graph of three independent experiments. Completed immunoblots are in . ( d ) Growth curves of HT29-EV and HT29-DUSP10 proliferative response treated with VP 1 µM and DMSO for 40 h after 2 h seeding (▼) using real-time proliferation analysis by xCELLigence technology. Linear regression analysis was performed (*** p < 0.001). Representative graph of three independent experiments.

Article Snippet: Human DUSP10 cDNA (SC31766, OriGene Technologies, Rockville, MD, USA) was subcloned in the pLenti-CMV/TO-Hygro vector using specific primers. pLenti-KO.1-Puro carrying specific shRNA sequences were used to knockdown DUSP10 constructs (shDUSP10).

Techniques: Expressing, Western Blot

Journal: Cancers

Article Title: DUSP10 Is a Regulator of YAP1 Activity Promoting Cell Proliferation and Colorectal Cancer Progression

doi: 10.3390/cancers11111767

Figure Lengend Snippet: DUSP10 interacts with YAP1 through Ser397 residue. ( a ) Expression of DUSP10, YAP1, p-YAP Ser397 , and p-YAP Ser127 proteins in DUSP10-wild type (DUSP10-WT), phosphatase catalytic site mutant (DUSP10-C408S), and p38 binding site mutant (DUSP10-AA) HT29 cell line in LD and HD. TUBULIN is the control protein. (Top) A representative image of three independent experiments. (Bottom) Quantification of all blots performed (mean ± SEM; Student’s t -test; * p < 0.05, ** p < 0.01, *** p < 0.001). ( b ) Analysis of DUSP10, YAP1, and p-YAP Ser127 proteins from nuclei and cytoplasm extracts of HT29 DUSP10 mutant cell lines in HD. LAMIN A/C and TUBULIN are used as nuclear and cytoplasm control proteins, respectively. (Left) A representative image of three independent experiments. (Right) Quantification of all blots performed (mean ± SEM; Student’s t -test; * p < 0.05, ** p < 0.01). ( c ) Immunoprecipitation of YAP-FLAG and detection of DUSP10 and p38 in HCT116. DUSP10-V5 and YAP-FLAG plasmids were co-transfected into the cell line and detected by anti-FLAG and anti-V5 antibodies, respectively. YAP1 wild type (YAP1-FLAG) and mutant (S381A-FLAG, S127A-FLAG) plasmids were immunoprecipitated with anti-FLAG. Representative images of three independent experiments. ( d ) Relative luciferase activity of the 8xGTII-luc (YAP/TEAD binding element reporter) was measured in HD, responding to DUSP10 overexpression and DUSP10 mutant constructs. HT29 (Top graph) and HCT116 (Bottom graph) were transiently transfected with the indicated plasmids and its control constructs. Student’s t -test (mean ± SEM; ** p < 0.01, *** p < 0.001) and three independent experiments were performed. Completed immunoblots of a–c are in , respectively.

Article Snippet: Human DUSP10 cDNA (SC31766, OriGene Technologies, Rockville, MD, USA) was subcloned in the pLenti-CMV/TO-Hygro vector using specific primers. pLenti-KO.1-Puro carrying specific shRNA sequences were used to knockdown DUSP10 constructs (shDUSP10).

Techniques: Expressing, Mutagenesis, Binding Assay, Immunoprecipitation, Transfection, Luciferase, Activity Assay, Over Expression, Construct, Western Blot

Journal: Cancers

Article Title: DUSP10 Is a Regulator of YAP1 Activity Promoting Cell Proliferation and Colorectal Cancer Progression

doi: 10.3390/cancers11111767

Figure Lengend Snippet: Effect of DUSP10 expression on Hippo-Salvador-Warts pathway in Drosophila melanogaster . ( a ) Subcellular localization of DUSP10-Myc (red) in salivary gland cells in sal EPv -Gal4 UAS-GFP/+; UAS-DUSP10-Myc/+ flies. ToPro3 (blue) was used as DNA marker and nuclei were detected by GFP (green) expression. Bars, 50 µm. ( b ) Higher magnification of a salivary gland cell from sal EPv -Gal4 UAS-GFP/+;UAS-DUSP10-Myc/+ individuals. Transversal section was represented. Bars, 5 µm. ( c ) Quantification of wing surfaces from anterior margin to L5 vein in wild type sal EPv -Gal4 UAS-GFP/+ (GFP) and sal EPv -Gal4/+;UAS-DUSP10-Myc/+ (DUSP10-Myc) individuals. Representative images of wing surfaces of both genotypes. Some cell clusters were detected between the two epithelial wings’ surfaces (red arrow). ( d ) Quantification of wing surfaces from margin to L5 vein in Ste -20 protein kinase hippo overexpressing alone ( sal EPv -Gal4 UAS-GFP/UAS-hpo, hpo/GFP) and combined to human DUSP10-Myc ( sal EPv -Gal4/UAS-hpo;UAS-DUSP10-Myc/+, hpo/DUSP10-Myc) individuals. Representative images of wing surfaces for both genotypes. ( e ) Quantification of wing surfaces from margin to L5 vein in Merlin/Nf2 knockdown alone ( sal EPv -Gal4 UAS-GFP/+;UAS-Mer-RNAi, Mer-RNAi/GFP) and combined to human DUSP10-Myc ( sal EPv -Gal4/UAS-Mer-RNAi;UAS-DUSP10-Myc/+, Mer-RNAi;DUSP10-Myc) individuals. Representative images of wing surfaces for both genotypes. All graphs were represented by the media ± standard deviation (SD) ( n = 10). Student’s t -test was performed to compare genotypes (** p < 0.01; *** p < 0.001).

Article Snippet: Human DUSP10 cDNA (SC31766, OriGene Technologies, Rockville, MD, USA) was subcloned in the pLenti-CMV/TO-Hygro vector using specific primers. pLenti-KO.1-Puro carrying specific shRNA sequences were used to knockdown DUSP10 constructs (shDUSP10).

Techniques: Expressing, Marker, Standard Deviation

Journal: Cancers

Article Title: DUSP10 Is a Regulator of YAP1 Activity Promoting Cell Proliferation and Colorectal Cancer Progression

doi: 10.3390/cancers11111767

Figure Lengend Snippet: DUSP10 is expressed in colon cancer patients and its nuclear expression is associated with a poor prognosis. ( a ) Representative images of DUSP10, p-p38, and YAP1 staining on a tissue microarray (TMA) in normal epithelial and tumor colon tissue from 73 human patients by immunohistochemistry (IHC). (Left) Image taken at 10× magnification (Bars, 100 μm) and (Right) magnified 400% by zoom. ( b ) Kaplan–Meier survival curve ( p < 0.05) of 999 colon cancer patients with weak (blue line) and strong (green line) nuclear DUSP10 expression in tumor epithelial tissue for six years.

Article Snippet: Human DUSP10 cDNA (SC31766, OriGene Technologies, Rockville, MD, USA) was subcloned in the pLenti-CMV/TO-Hygro vector using specific primers. pLenti-KO.1-Puro carrying specific shRNA sequences were used to knockdown DUSP10 constructs (shDUSP10).

Techniques: Expressing, Staining, Microarray, Immunohistochemistry

Journal: Cancers

Article Title: DUSP10 Is a Regulator of YAP1 Activity Promoting Cell Proliferation and Colorectal Cancer Progression

doi: 10.3390/cancers11111767

Figure Lengend Snippet: Analysis of tumor epithelial tissue from colon cancer patients according to protein expression. COX2, DUSP10, YAP1, and p-p38 expression are analyzed in tumor epithelial tissue from a cohort of 73 colon cancer patients. Two-way ANOVA analysis was performed.

Article Snippet: Human DUSP10 cDNA (SC31766, OriGene Technologies, Rockville, MD, USA) was subcloned in the pLenti-CMV/TO-Hygro vector using specific primers. pLenti-KO.1-Puro carrying specific shRNA sequences were used to knockdown DUSP10 constructs (shDUSP10).

Techniques: Expressing, Staining

Journal: Cancers

Article Title: DUSP10 Is a Regulator of YAP1 Activity Promoting Cell Proliferation and Colorectal Cancer Progression

doi: 10.3390/cancers11111767

Figure Lengend Snippet: Analysis of DUSP10 expression in colon cancer tissue according to clinical-pathological categories. DUSP10 expression was analyzed in tumor epithelial tissue from a cohort of 999 patients depending on clinical-pathological categories (sex, Dukes’ stage, grade of differentiation, origin, or patient’s status). Two-way ANOVA analysis was performed to study the patient variation.

Article Snippet: Human DUSP10 cDNA (SC31766, OriGene Technologies, Rockville, MD, USA) was subcloned in the pLenti-CMV/TO-Hygro vector using specific primers. pLenti-KO.1-Puro carrying specific shRNA sequences were used to knockdown DUSP10 constructs (shDUSP10).

Techniques: Expressing

Journal: Cancers

Article Title: DUSP10 Is a Regulator of YAP1 Activity Promoting Cell Proliferation and Colorectal Cancer Progression

doi: 10.3390/cancers11111767

Figure Lengend Snippet: Survival analysis of colon cancer patients depending on nuclear DUSP10 protein expression. A cohort of 999 colon cancer patients was analyzed and classified by weak and strong staining for DUSP10. Number of events (deaths per group) were quantified. The table represents the relative hazard ratio (RR) and 95% confidence interval (CI).

Article Snippet: Human DUSP10 cDNA (SC31766, OriGene Technologies, Rockville, MD, USA) was subcloned in the pLenti-CMV/TO-Hygro vector using specific primers. pLenti-KO.1-Puro carrying specific shRNA sequences were used to knockdown DUSP10 constructs (shDUSP10).

Techniques: Expressing, Staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: Oxidative stress-induced JNK activation contributes to proinflammatory phenotype of aging diabetic mesangial cells

doi: 10.1152/ajprenal.00078.2009

Figure Lengend Snippet: Decreased mitogen-activated protein kinase phosphatase 5 (MKP5) mRNA expression in glomeruli and mesangial cells of OD mice. Glomeruli were microdissected from OD and O mice (n = 10/group). MKP5 mRNA expression was determined by a real-time PCR. A: glomeruli. **P < 0.01 vs. glomeruli of O mice. B: mesangial cells were isolated from 2 separate mice of Y, YD, O, and OD, respectively. *P < 0.05 vs. mesangial cells from young controls (n = 2 cell lines/age). ##P < 0.01 vs. mesangial cells from old controls (n = 2 cell lines/group).

Article Snippet: Old diabetic mesangial cells were transfected with a pCMV6 entry plasmid containing a full-length human MKP5 cDNA and a G418 resistant site (Origene, Rockville, MD).

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

Journal: American Journal of Physiology - Renal Physiology

Article Title: Oxidative stress-induced JNK activation contributes to proinflammatory phenotype of aging diabetic mesangial cells

doi: 10.1152/ajprenal.00078.2009

Figure Lengend Snippet: Overexpression of MKP5 in OD/MC was associated with decreased phosphorylated JNK and inflammation. A: 3 MKP5 stably transfected OD/MC lines showed increased MKP5 mRNA expression as determined by real-time PCR. The level in untransfected OD/MC was arbitrarily defined as 1. B: phosphorylated JNK levels were decreased in 3 MKP5 stably transfected OD/MC lines (OD/MC + MKP5). The reduction was more obvious in lane 2 and 3. C–E: all 3 MKP5 stably transfected OD/MC showed decreased MCP-1, CXCL-1, and MIP2 mRNA expression compared with untransfected cells or cells transfected with an empty vector. *P < 0.05, **P < 0.01 vs. OD/MC transfected with an empty vector (n = 3).

Article Snippet: Old diabetic mesangial cells were transfected with a pCMV6 entry plasmid containing a full-length human MKP5 cDNA and a G418 resistant site (Origene, Rockville, MD).

Techniques: Over Expression, Stable Transfection, Transfection, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: American Journal of Physiology - Renal Physiology

Article Title: Oxidative stress-induced JNK activation contributes to proinflammatory phenotype of aging diabetic mesangial cells

doi: 10.1152/ajprenal.00078.2009

Figure Lengend Snippet: Knocking down of MKP5 expression in O/MC was associated with increased JNK activity and inflammation. A: 2 lines of mesangial cells from O/MC were transfected with MKP5 siRNA for 48 h. MKP5 mRNA levels were determined by real-time PCR. The level in O/MC was arbitrarily defined as 1. B: representative gel of JNK kinase activity and phosphorylated JNK level in O/MC with or without MKP5 knockdown. JNK activity, reflected by the levels of phosphorylated c-Jun on the top of the gel, was increased in O/MC with MKP5 knockdown. Knocking down of MKP5 also increased phosphorylated JNK levels in these cells while the levels of total JNK and β-actin remained similar between cells with and without MKP5 knockdown. C: MCP-1 production was increased in O/MC with MKP5 knockdown (n = 2). *P < 0.05 vs. O/MC (n = 2).

Article Snippet: Old diabetic mesangial cells were transfected with a pCMV6 entry plasmid containing a full-length human MKP5 cDNA and a G418 resistant site (Origene, Rockville, MD).

Techniques: Expressing, Activity Assay, Transfection, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Renal Physiology

Article Title: Oxidative stress-induced JNK activation contributes to proinflammatory phenotype of aging diabetic mesangial cells

doi: 10.1152/ajprenal.00078.2009

Figure Lengend Snippet: H2O2 decreased MKP5 phosphatase activity. Enzymatic active recombinant MKP5 was incubated with or without 5 mM H2O2 in the presence or absence of dithiothreitol (DTT; 5 mM) for 30 min. Results shown were from 3 separate measurements of MKP5 phosphatase activity using 3-O-methylfluorescein as a substrate. The activity in untreated cells was arbitrarily defined as 100%.

Article Snippet: Old diabetic mesangial cells were transfected with a pCMV6 entry plasmid containing a full-length human MKP5 cDNA and a G418 resistant site (Origene, Rockville, MD).

Techniques: Activity Assay, Recombinant, Incubation