Journal: Biomarker Research

Article Title: Identification of GRIN2D as a novel therapeutic target in pancreatic ductal adenocarcinoma

doi: 10.1186/s40364-023-00514-4

Figure Lengend Snippet: GRIN2D was identified as an oncogene in PDAC. ( A ). Workflow of in-vivo genome-wide RNAi screening. ( B ). GRIN2D was upregulated in PDAC cells, as compared to non-tumor HPDE cells. ( C ). IHC staining of GRIN2D in blood vessels and ducts with the respective markers PECAM-1 and CK19. GRIN2D was upregulated in PDAC tumor tissues, compared to adjacent non- tumor tissues. ( D ). GRIN2D expression was increased in PDAC tumors, as compared to normal, from two independent samples cohort GSE16515 and GSE15471. Data are from at least three independent experiments. Mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: PDAC cell line Capan2 cells were transduced with lentivirus carrying a human genome-wide shRNA library (GeneNetTM Human 50 K siRNA Library, System Biosciences).

Techniques: In Vivo, Genome Wide, Immunohistochemistry, Expressing

Journal: Biomarker Research

Article Title: Identification of GRIN2D as a novel therapeutic target in pancreatic ductal adenocarcinoma

doi: 10.1186/s40364-023-00514-4

Figure Lengend Snippet: GRIN2D drove tumorigenic functions in PDAC cells. ( A ). GRIN2D was knocked down by siRNAs in SW1990 and PANC04.03 cells. ( B ). Knockdown of GRIN2D inhibited cell growth in PDAC cells. ( C ). Knockdown of GRIN2D inhibited colony formation in PDAC cells. ( D ). Knockdown of GRIN2D inhibited cell migration in PDAC cells. ( E ). Knockdown of GRIN2D inhibited cell invasion in PDAC cells. Cells in the colony formation assay and invasion assay were stained by crystal violet. Data are from at least three independent experiments. Mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: PDAC cell line Capan2 cells were transduced with lentivirus carrying a human genome-wide shRNA library (GeneNetTM Human 50 K siRNA Library, System Biosciences).

Techniques: Knockdown, Migration, Colony Assay, Invasion Assay, Staining

Journal: Biomarker Research

Article Title: Identification of GRIN2D as a novel therapeutic target in pancreatic ductal adenocarcinoma

doi: 10.1186/s40364-023-00514-4

Figure Lengend Snippet: GRIN2D promotes PDAC progression by CREB/ p38 MAPK signaling pathway. ( A ). p38, MSK, and CREB were dephosphorylated after knockdown of GRIN2D in SW1990 and PANC04.03 cells. ( B ). Clinical correlation between GRIN2D and p-CREB levels in PDAC primary tumors. ( C ). Amount of common differential genes in SW1990 and PANC04.03 cells after knockdown of GRIN2D. ( D ). Expression of differential genes after knockdown of GRIN2D. ( E ). Correlation between GRIN2D and IL20RB, CCL22, G5S2, IL6, EPGN, and HMGA2 expression in PDAC tumors. ( F ). IL20RB, CCL22, G5S2, IL6, EPGN, and HMGA2 expression in tumor and non- tumor groups from two independent samples cohort GSE16515 and GSE15471. ( G ). Survival analysis of IL20RB, CCL22, G5S2, IL6, EPGN, and HMGA2 in PDAC patients. ( H ). Phosphorylated CREB binding to HMGA2 and IL20RB in SW1990 and PANC04.03 cells, as revealed by ChIP assay. ( I ). EMT markers ZEB1, SNAIL, SLUG, and TWIST were downregulated after knockdown of GRIN2D in SW1990 and PANC04.03 cells. Data are from at least three independent experiments. Mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: PDAC cell line Capan2 cells were transduced with lentivirus carrying a human genome-wide shRNA library (GeneNetTM Human 50 K siRNA Library, System Biosciences).

Techniques: Knockdown, Expressing, Binding Assay

Journal: Biomarker Research

Article Title: Identification of GRIN2D as a novel therapeutic target in pancreatic ductal adenocarcinoma

doi: 10.1186/s40364-023-00514-4

Figure Lengend Snippet: GRIN2D promoted tumor growth and liver metastasis in PDAC. ( A ). GRIN2D was knocked down by stable lentiviral shRNA system in SW1990 cells. ( B ). Knockdown of GRIN2D inhibited tumor growth in SW1990 cells. Photographs of mice xenograft at day 28 after knockdown of GRIN2D in SW1990 cells. ( C ). Tumor volume and tumor mass of xenograft mice after knockdown of GRIN2D. ( D ). IHC staining of Ki67, H&E Staining in mice subcutaneous tumors after knockdown of GRIN2D. ( E ). Knockdown of GRIN2D inhibited liver metastasis in SW1990 cells

Article Snippet: PDAC cell line Capan2 cells were transduced with lentivirus carrying a human genome-wide shRNA library (GeneNetTM Human 50 K siRNA Library, System Biosciences).

Techniques: shRNA, Knockdown, Immunohistochemistry, Staining

Journal: Biomarker Research

Article Title: Identification of GRIN2D as a novel therapeutic target in pancreatic ductal adenocarcinoma

doi: 10.1186/s40364-023-00514-4

Figure Lengend Snippet: Memantine is a potential drug for PDAC therapy. ( A ). Dose-response curve of memantine in PDAC cells. ( B ). Cell growth was inhibited in memantine treated PDAC cells, as revealed by CCk8 cell viability assay. ( C ). Cell migration was inhibited in memantine treated PDAC cells, as revealed by wound healing cell migration assay. ( D ). Clonogenic ability was inhibited in memantine treated PDAC cells, the cells in the colony formation assay were stained by crystal violet. ( E ). Level of GRIN2D decreased in memantine treated PDAC cells. ( F ). p38, MSK, and CREB in p38 MAPK signaling pathway were dephosphorylated after treatment of memantine. Data are from at least three independent experiments. Mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: PDAC cell line Capan2 cells were transduced with lentivirus carrying a human genome-wide shRNA library (GeneNetTM Human 50 K siRNA Library, System Biosciences).

Techniques: Viability Assay, Migration, Cell Migration Assay, Colony Assay, Staining

Journal: Protein & Cell

Article Title: Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation

doi: 10.1007/s13238-020-00760-4

Figure Lengend Snippet: HNF4G is an important player in PDAC progression and invasiveness. (A) Scheme of reanalyzing PDAC susceptibility genes using combined GWAS data. (B) High content screening strategy for 36 candidate genes in PDAC cells. Scale bar: 100 μm. (C) Heatmap showing the results of high content screening with a siRNAs library in PDAC cells. (D) The effect of siRNA knockdown of indicated genes on migration ability of PANC-1 cells. Data represent mean ± SEM from 3 experiments. (E) Immunohistochemical (IHC) staining of HNF4G in tissue array consisting of 65 PDAC samples. Left panel, representative IHC images, Scale bar: 700 μm (left images) and 60 μm (right images); right panel, quantification statistic. (F) Scatter dot plots showing HNF4G expression levels in PDAC tumor and normal samples. Data are derived from the Gene Expression Profiling Interactive Analysis (GEPIA). (G) Scatter dot plots showing HNF4G expression levels in early and latter stage of PDAC. Data were derived from the TCGA PDAC dataset. (H) Kaplan-Meier plots of overall survival of patients derived from the TCGA PDAC dataset stratified by HNF4G expression. The best performing threshold is used as a cutoff. HR, hazard ratio; CI, confidence interval. Statistical significance: *, P < 0.05, **, P < 0.01 and ****, P < 0.0001 of Student’s t -test or Wilcoxon rank-sum test. ns, not significant

Article Snippet: PDAC cell lines BxPC-3, CFPAC-1, PANC-1, Capan-2 and MIAPaCa-2 were obtained from the China Center for Type Culture Collection while AsPC-1 and T3M4 were kind gifts from Dr. G. Yang (PUMCH).

Techniques: High Content Screening, Knockdown, Migration, Immunohistochemical staining, Immunohistochemistry, Expressing, Derivative Assay, Gene Expression

Journal: Protein & Cell

Article Title: Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation

doi: 10.1007/s13238-020-00760-4

Figure Lengend Snippet: HNF4G upregulation is caused by SMAD4 deficiency in PDAC. (A) HNF4G mRNA levels in PDAC as function of SMAD4 copy-number variation. Data were derived from the TCGA database. (B) The relationship between HNF4G and SMAD4 protein levels in PDAC determined by IHC staining. Left panel shows representative IHC images of HNF4G and SMAD4 in serial sections of PDAC tissue array ( n = 185). Scale bar in left images = 600 μm. Scale bar in right images = 200 μm. Right panel shows HNF4G levels as function of SMAD4 levels both expressed as IHC scores: low, 0; medium, 1–4; and high, 6–12. (C) The expression levels of HNF4G RNA (left) and protein (right) in 4 PDAC cell lines with SMAD4 deficiency and 3 cell lines without SMAD4 deficiency. Data represent mean ± SEM from 3 independent determinations and each had triplicates. (D and E) The effects of SMAD4 knockdown (D) or overexpression (E) on HNF4G RNA (upper panel) and protein levels (lower panel) in PDAC cells. Data are mean ± SEM from 3 independent determinations and each had triplicates. (F and G) Relative expression levels of reporter gene bearing the HNF4G promoter region in T3M4 cells with or without SMAD4 overexpression (F) and in MIAPaCa-2 cells with or without SMAD4 knockdown (G). (H) Relative expression levels of reporter gene bearing the mutated HNF4G promoter region in PDAC cells. Each promoter harbors a mutated SBE. Mutation in SBE 1 had the most significant impact on reporter gene expression compared with vector control and the mutation in other SBEs. Results are mean ± SEM from 3 experiments and each had 6 replicates. (I and J) Chromatin immunoprecipitation assays showing binding of SMAD4 to HNF4G promoter region SBE 1 in PDAC cells (I) and knockdown of SMAD4 expression in these cells substantially decreased the binding (J). Fold enrichment represents DNA levels associated with HNF4G or IgG (as control) relative to an input control from 3 independent experiments. Data are mean ± SEM of 3 experiments. Statistical significance: *, P < 0.05, **, P < 0.01, ***, P < 0.001 and ****, P < 0.0001 of Student’s t -test, χ 2 test or Wilcoxon rank-sum test. ns, not significant

Article Snippet: PDAC cell lines BxPC-3, CFPAC-1, PANC-1, Capan-2 and MIAPaCa-2 were obtained from the China Center for Type Culture Collection while AsPC-1 and T3M4 were kind gifts from Dr. G. Yang (PUMCH).

Techniques: Derivative Assay, Immunohistochemistry, Expressing, Knockdown, Over Expression, Mutagenesis, Gene Expression, Plasmid Preparation, Control, Chromatin Immunoprecipitation, Binding Assay

Journal: Protein & Cell

Article Title: Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation

doi: 10.1007/s13238-020-00760-4

Figure Lengend Snippet: HNF4G overexpression promotes PDAC cell invasiveness and activates the cell-cell junction pathway. (A) HNF4G overexpression promoted migration and invasion of PDAC cells in vitro . Left panel shows representative images of transwell assays and right panel shows quantification statistic. Data are mean ± SEM from 3 independent experiments and each had duplicate. (B) HNF4G overexpression promoted migration and invasion of PDAC cells transplanted in the pancreas of mice ( n = 3). Left panel shows representative bioluminescence images taken at 7 and 40 days of implantation; Right panel shows quantitative fluorescent intensity of the transplanted PDAC. (C) Representative H&E staining pictures of the pancreas, liver and lung from mice implanted orthotopically with PDAC cells with or without HNF4G overexpression. Scale bars: 100 μm. (D) The work flow schematic for analyzing the candidate genes targeted by HNF4G. (E) Functional enrichment of the 293 HNF4G-targeted genes by Metascape. (F) The expression levels of some downstream genes of HNF4G in PDAC cells with or without HNF4G overexpression. Results are mean ± SEM from 3 independent determinations and each had triplicate. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001 and ****, P < 0.0001 of Student’s t -test or Wilcoxon rank-sum test. ns, not significant

Article Snippet: PDAC cell lines BxPC-3, CFPAC-1, PANC-1, Capan-2 and MIAPaCa-2 were obtained from the China Center for Type Culture Collection while AsPC-1 and T3M4 were kind gifts from Dr. G. Yang (PUMCH).

Techniques: Over Expression, Migration, In Vitro, Staining, Functional Assay, Expressing

Journal: Protein & Cell

Article Title: Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation

doi: 10.1007/s13238-020-00760-4

Figure Lengend Snippet: Metformin activates AMPK that induces HNF4G phosphorylation-ubiquitination coupled degradation. (A) Effect of Metformin (10 μmol/L) on HNF4G and AMPKα phosphorylation in T3M4 cells. (B) Metformin (10 μmol/L) promoted AMPKα phosphorylation and HNF4G degradation in T3M4 cells. (C) Metformin promoted HNF4G degradation but not inhibited its synthesis in T3M4 cells. Left panel, Metformin treatment substantially decreased the HNF4G levels with time in cells exposed to protein synthesis inhibitor cycloheximide (CHX; 20 μg/mL) compared with cells exposed to vehicle; right panel, Metformin treatment no longer substantially decreased HNF4G level in cells exposed to proteasome inhibitor MG132 (5 μmol/L). (D) Metformin promotes HNF4G ubiquitination. T3M4 cells were treated with or without Metformin (10 μmol/L). Cell lysates were immunoprecipitated (IP) with either control IgG or antibody against HNF4G and analyzed by immunoblotting with a ubiquitin (Ub)-specific antibody. Bottom panels, input from cell lysates. (E) Immunoblot analysis of HNF4G phosphorylation status in T3M4 cells with or without AMPKα knockdown treated with Metformin (10 μmol/L). (F and G) Immunoblot analysis of phosphorylated HNF4G and AMPKα in T3M4 cells, cells with HNF4G knockout or cells with ectopic expression of S382A-mutated HNF4G exposed to Metformin (10 μmol/L) for different times. (H) Metformin treatment significantly decreased the expression levels of some oncogenes in PDAC cells compared with vehicle controls. Results are mean ± SEM from 3 independent determinations and each had triplicate. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 of Student’s t -test

Article Snippet: PDAC cell lines BxPC-3, CFPAC-1, PANC-1, Capan-2 and MIAPaCa-2 were obtained from the China Center for Type Culture Collection while AsPC-1 and T3M4 were kind gifts from Dr. G. Yang (PUMCH).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Immunoprecipitation, Control, Western Blot, Knockdown, Knock-Out, Expressing

Journal: Protein & Cell

Article Title: Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation

doi: 10.1007/s13238-020-00760-4

Figure Lengend Snippet: Metformin suppress HNF4G-induced PDAC metastasis depending on SMAD4 status. (A) Metformin treatment significantly repressed in vitro migration and invasion of SMAD4-deficient T3M4 cells but not SMAD4-efficient PANC-1. Left panels show representative images of transwell assays and right panels represent quantitative statistic. Data are mean ± SEM from 3 independent experiments and each had triplicate. Shown are the results in cells treated with or without Metformin (10 μmol/L); see also Fig. S5 for the entire and detailed dose-dependent results. (B) Knockdown of SMAD4 expression significantly promoted Metformin to suppress the migration and invasion of PDAC cells. Upper panel are representative transwell images and lower panel are quantitative data (mean ± SEM from 3 independent experiments and each had triplicate). (C) Metformin treatment significantly repressed the spread of HNF4G-overexpressing T3M4 cells implanted in mouse pancreas ( n = 4). Left panel shows bioluminescence images of mice and the right panel shows quantitative fluorescent intensities. (D) Metformin treatment significantly prolonged survival time of mice implanted with PDAC in the pancreas as compared with vehicle control. (E) Metformin treatment significantly reduced PDAC metastases in the liver compared with vehicle control. Left panel shows representative tumor nodes staining by H&E and right panel shows quantitative statistic. Scale bars: 100 μm (40×) and 10 μm (400×). (F) Representative IHC pictures showing that Metformin treatment substantially reduced HNF4G but increased p-AMPKα expression levels in serial sections of both pancreas and liver from mice with PDAC implantation as compared with vehicle control. Scale bars: 100 μm (100×) and 25 μm (400×). Statistical significance: **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 of Student’s t -test or Wilcoxon rank-sum test. ns, not significant

Article Snippet: PDAC cell lines BxPC-3, CFPAC-1, PANC-1, Capan-2 and MIAPaCa-2 were obtained from the China Center for Type Culture Collection while AsPC-1 and T3M4 were kind gifts from Dr. G. Yang (PUMCH).

Techniques: In Vitro, Migration, Knockdown, Expressing, Control, Staining

Journal: Protein & Cell

Article Title: Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation

doi: 10.1007/s13238-020-00760-4

Figure Lengend Snippet: Metformin treatment improves clinical outcomes of patients with SMAD4-deficient PDAC. (A) Representative images of IHC staining of SMAD4 and HNF4G proteins in PDAC. Scale bar = 100 μm. (B) The correlation between HNF4G and SMAD4 protein levels in PDAC determined by IHC score. SMAD4−, IHC score = 0; SMAD4+, IHC score > 0. HNF4G Low level, scores 0–4; HNF4G high level, scores 6–12. (C) The distribution of patients by PDAC tumor stage and SMAD4 status as function of Metformin treatment. (D) Kaplan-Meier estimate of survival time in 118 patients with PDAC by SMAD4 status and Metformin treatment. Hazard ratio (HR) and 95% confidence interval (CI) were calculated with age, sex, tumor stage as covariates. SMAD4−, IHC score = 0; SMAD4+, IHC score > 0. Statistical significance: *, P < 0.05 and**, P < 0.01 of χ 2 test. ns, not significant

Article Snippet: PDAC cell lines BxPC-3, CFPAC-1, PANC-1, Capan-2 and MIAPaCa-2 were obtained from the China Center for Type Culture Collection while AsPC-1 and T3M4 were kind gifts from Dr. G. Yang (PUMCH).

Techniques: Immunohistochemistry

Journal: Protein & Cell

Article Title: Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation

doi: 10.1007/s13238-020-00760-4

Figure Lengend Snippet: A proposed working model for aberrant SMAD4-HNF4G in PDAC invasiveness and the effect of Metformin. In PDAC cells where SMAD4 is sufficient, the expression of the downstream oncogene HNF4G that promotes PDAC invasiveness is physiologically inhibited by the SMAD complex. In PDAC cells where SMAD4 is deficient, the expression of HNF4G is over-activated, which evokes cancer cell invasion and metastasis. Metformin may act as a target drug repressing PDAC invasion and metastasis by activating AMPK that induces phosphorylation-ubiquitination coupled HNF4G degradation

Article Snippet: PDAC cell lines BxPC-3, CFPAC-1, PANC-1, Capan-2 and MIAPaCa-2 were obtained from the China Center for Type Culture Collection while AsPC-1 and T3M4 were kind gifts from Dr. G. Yang (PUMCH).

Techniques: Expressing, Phospho-proteomics, Ubiquitin Proteomics

Journal: Cancer Science

Article Title: Deubiquitinase ubiquitin‐specific peptidase 10 maintains cysteine rich angiogenic inducer 61 expression via Yes1 associated transcriptional regulator to augment immune escape and metastasis of pancreatic adenocarcinoma

doi: 10.1111/cas.15326

Figure Lengend Snippet: Ubiquitin‐specific peptidase 10 (USP10) is abundantly expressed in pancreatic adenocarcinoma (PAAD) tissues signaling pathway enriched on the positive side of the YAP1 according to Gene Set Enrichment Analysis (GSEA and cells and positively correlated with YAP1. (A) Hydrolysis‐related); (B) genes show high correlation with YAP1 analyzed using a correlation test (cor. Test); (C) USP10 expression in TCGA‐PAAD and GTEx databases; (D) association between USP10 level and the prognosis of patients analyzed in TCGA‐PAAD; (E) staining intensity of USP10 in the PAAD tissues and normal pancreatic tissues in the HPA system; (F and G) expression of Cyr61 (F) and CTGF (G) in TCGA‐PAAD; (H and I) positive correlations between CTGF (H) and Cyr61 (I), and USP10 examined by Spearman's rank correlation coefficient; (J) expression of USP10 in PAAD cell lines (Capan‐2, AsPC‐1, Panc‐1, and BxPC‐1), and in the normal HPDE6 cells determined by RT‐qPCR. Differences were analyzed by one‐way ANOVA (J), *** P < 0.001

Article Snippet: A human pancreatic duct epithelial cell line HPDE6 and PAAD cell lines (Capan‐2, Panc‐1, AsPC‐1, and BxPC‐1) were purchased from Genechem.

Techniques: Ubiquitin Proteomics, Expressing, Staining, Quantitative RT-PCR

Journal: Cancer Science

Article Title: Deubiquitinase ubiquitin‐specific peptidase 10 maintains cysteine rich angiogenic inducer 61 expression via Yes1 associated transcriptional regulator to augment immune escape and metastasis of pancreatic adenocarcinoma

doi: 10.1111/cas.15326

Figure Lengend Snippet: Ubiquitin‐specific peptidase 10 (USP10) knockdown increases ubiquitination and degradation of YAP1 in pancreatic adenocarcinoma (PAAD) cells. (A) Protein levels of YAP1, Cyr61, and USP10 in Panc‐1 and BxPC‐1 cells after shUSP10 transfection examined by western blot analysis; (B) binding relationship between USP10 and YAP1 in Panc‐1 and BxPC‐1 cells examined by the Co‐IP assay; (C) sub‐cellular distribution of USP10 and YAP1 in Panc‐1 and BxPC‐1 cells examined using dual immunofluorescence staining; (D) ubiquitination level of YAP1 in 293T cells after HA‐ubiquitin, Flag‐YAP1, or USP10 overexpression; (E) ubiquitination level of YAP1 in Panc‐1 and BxPC3 cells after USP10 knockdown examined by Co‐IP; and (F) protein level of YAP1 in Panc‐1 and BxPC3 cells after 10 μM MG132 treatment determined by western blot analysis. Differences were analyzed by two‐way ANOVA (A), ** P < 0.01

Article Snippet: A human pancreatic duct epithelial cell line HPDE6 and PAAD cell lines (Capan‐2, Panc‐1, AsPC‐1, and BxPC‐1) were purchased from Genechem.

Techniques: Ubiquitin Proteomics, Knockdown, Transfection, Western Blot, Binding Assay, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Over Expression

Journal: Cancer Science

Article Title: Deubiquitinase ubiquitin‐specific peptidase 10 maintains cysteine rich angiogenic inducer 61 expression via Yes1 associated transcriptional regulator to augment immune escape and metastasis of pancreatic adenocarcinoma

doi: 10.1111/cas.15326

Figure Lengend Snippet: Ubiquitin‐specific peptidase 10 (USP10) knockdown reduces the immune escape of pancreatic adenocarcinoma (PAAD) cells. (A and B) expression of PD‐L1 and Galectin‐9 in Panc‐1 and BxPC‐1 cells detected by RT‐qPCR and western blot analysis; (C) portion of programmed death ligand‐1 (PD‐L1)‐ and Galectin‐9‐positive Panc‐1 and BxPC‐1 cells examined by flow cytometry; (D) portion of CD86‐positive (M1‐polarized) and CD206‐positive (M2‐polarized) macrophages after co‐incubation with Panc‐1 and BxPC‐1 cells evaluated by flow cytometry; (E) expression of the M1‐macrophage markers (iNOS, IL‐1β, and IL‐6) and M2‐macrophage markers (Arg1, IL‐10, and IL‐13) in cells examined by RT‐qPCR; (F) the degree of Panc‐1 and BxPC‐1 cells lysed by natural killer (NK) cells examined using an LDH kit. Differences were analyzed by two‐way ANOVA (A–E), ** P < 0.01; *** P < 0.001

Article Snippet: A human pancreatic duct epithelial cell line HPDE6 and PAAD cell lines (Capan‐2, Panc‐1, AsPC‐1, and BxPC‐1) were purchased from Genechem.

Techniques: Ubiquitin Proteomics, Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Incubation

Journal: Cancer Science

Article Title: Deubiquitinase ubiquitin‐specific peptidase 10 maintains cysteine rich angiogenic inducer 61 expression via Yes1 associated transcriptional regulator to augment immune escape and metastasis of pancreatic adenocarcinoma

doi: 10.1111/cas.15326

Figure Lengend Snippet: Ubiquitin‐specific peptidase 10 (USP10) knockdown reduces growth and invasiveness of pancreatic adenocarcinoma (PAAD) cells in vitro . (A) viability of Panc‐1 and BxPC‐1 cells examined using a CTG kit; (B) proliferation rate of the Panc‐1 and BxPC‐1 cells using the EdU labeling kit; (C) apoptosis of Panc‐1 and BxPC‐1 cells examined using flow cytometry; (D and E) levels of EMT‐related biomarkers ZO‐1, E‐cadherin, Vimentin, and N‐cadherin in Panc‐1 and BxPC‐1 cells determined by RT‐qPCR (D), western blot analysis (E), and immunofluorescence staining (F), respectively; (G and H) migration (G) and invasion (H) ability of Panc‐1 and BxPC‐1 cells determined by Transwell assays. Differences were analyzed by two‐way ANOVA (A–E), ** P < 0.01; *** P < 0.001

Article Snippet: A human pancreatic duct epithelial cell line HPDE6 and PAAD cell lines (Capan‐2, Panc‐1, AsPC‐1, and BxPC‐1) were purchased from Genechem.

Techniques: Ubiquitin Proteomics, Knockdown, In Vitro, Labeling, Flow Cytometry, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Migration

Journal: Cancer Science

Article Title: Deubiquitinase ubiquitin‐specific peptidase 10 maintains cysteine rich angiogenic inducer 61 expression via Yes1 associated transcriptional regulator to augment immune escape and metastasis of pancreatic adenocarcinoma

doi: 10.1111/cas.15326

Figure Lengend Snippet: Ubiquitin‐specific peptidase 10 (USP10) knockdown suppresses growth and metastasis of pancreatic adenocarcinoma (PAAD) cells in vivo. (A) Growth rate of the xenograft tumors; (B) weight of the xenograft tumors on the 35th day; (C–G) staining intensity of KI67 (C), PCNA (D), YAP1 (E), Cyr61 (F), and CTGF (G) in the tissues of xenograft tumors detected by IHC staining; and (H and I) tumor metastasis in murine lung (H) and liver (I) tissues on the 45th day examined by HE staining. In each group, n = 6. Differences were analyzed by two‐way ANOVA (A–E), ** P < 0.01; *** P < 0.001

Article Snippet: A human pancreatic duct epithelial cell line HPDE6 and PAAD cell lines (Capan‐2, Panc‐1, AsPC‐1, and BxPC‐1) were purchased from Genechem.

Techniques: Ubiquitin Proteomics, Knockdown, In Vivo, Staining, Immunohistochemistry

Journal: Cancer Science

Article Title: Deubiquitinase ubiquitin‐specific peptidase 10 maintains cysteine rich angiogenic inducer 61 expression via Yes1 associated transcriptional regulator to augment immune escape and metastasis of pancreatic adenocarcinoma

doi: 10.1111/cas.15326

Figure Lengend Snippet: Overexpression of Cyr61 restores immune tolerance of pancreatic adenocarcinoma (PAAD) cells. (A) Protein level of Cyr61 in Panc‐1 and BxPC‐1 cells after oe‐Cyr61 transfection examined by western blot analysis; (B) PD‐L1 and Galectin‐9 expression in Panc‐1 and BxPC‐1 cells examined by RT‐qPCR; (C) portion of PD‐L1‐ and Galectin‐9‐positive cells evaluated by the flow cytometry; (D) portion of CD86‐positive (M1‐polarized) and CD206‐positive (M2‐polarized) macrophages after co‐incubation with Panc‐1 and BxPC‐1 cells detected by flow cytometry; (E) expression of the M1‐macrophage markers (iNOS, IL‐1β, and IL‐6) and M2‐macrophage markers (Arg1, IL‐10, and IL‐13) in cells examined by RT‐qPCR; (F) the degree of Panc‐1 and BxPC‐1 cells lysed by natural killer (NK) cells examined using an LDH kit. Differences were analyzed by two‐way ANOVA (A–E), ** P < 0.01

Article Snippet: A human pancreatic duct epithelial cell line HPDE6 and PAAD cell lines (Capan‐2, Panc‐1, AsPC‐1, and BxPC‐1) were purchased from Genechem.

Techniques: Over Expression, Transfection, Western Blot, Expressing, Quantitative RT-PCR, Flow Cytometry, Incubation

Journal: Cancer Science

Article Title: Deubiquitinase ubiquitin‐specific peptidase 10 maintains cysteine rich angiogenic inducer 61 expression via Yes1 associated transcriptional regulator to augment immune escape and metastasis of pancreatic adenocarcinoma

doi: 10.1111/cas.15326

Figure Lengend Snippet: Overexpression of Cyr61 restores pancreatic adenocarcinoma (PAAD) cell proliferation in vitro. (A) Viability of Panc‐1 and BxPC‐1 cells examined using a CellTiter Glo (CTG) kit; (B) proliferation rate of the Panc‐1 and BxPC‐1 cells using the EdU labeling kit; (C) apoptosis of Panc‐1 and BxPC‐1 cells examined using flow cytometry; (D–F) levels of EMT‐related biomarkers ZO‐1, E‐cadherin, Vimentin, and N‐cadherin in Panc‐1 and BxPC‐1 cells determined by RT‐qPCR (D), western blot analysis (E), and immunofluorescence staining (F), respectively; and (G and H) migration (G) and invasion (H) ability of Panc‐1 and BxPC‐1 cells determined by Transwell assays. Differences were analyzed by two‐way ANOVA (A–G), ** P < 0.01

Article Snippet: A human pancreatic duct epithelial cell line HPDE6 and PAAD cell lines (Capan‐2, Panc‐1, AsPC‐1, and BxPC‐1) were purchased from Genechem.

Techniques: Over Expression, In Vitro, Labeling, Flow Cytometry, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Migration