Journal: Cell reports. Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy.

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet: Figure 1. Secreted AGR2 promotes pancreatic carcinogenesis by activating CAFs (A) Representative immunohistochemical (IHC) images demonstrate AGR2-positive and -negative PDAC tumors (scale bars: 50 mm). (B) Survival curves of patients categorized by AGR2 expression in PDAC tumor samples via IHC (n = 99). (C) Survival curves of patients stratified according to median AGR2 levels, measured by ELISA, in serum from individuals with PDAC (n = 172). (D) Western blot analyses of AGR2 expression in human PDAC cell lines (Capan2 and Panc1) following CRISPR-Cas9-mediated AGR2 knockout (performed in triplicate). (E) Images and volumes of control versus AGR2-knockout subcutaneous xenografts derived from Capan2 and Panc1 cell lines in nude mice (n = 5 per group). (F) Subcutaneous xenografts from AGR2-knockout Capan2 and Panc1 cells (AGR2KO), following re-expression of wild-type AGR2 (AGR2WT), AGR2 lacking a nuclear localization signal (AGR2DNLS), and AGR2 lacking a signal peptide (AGR2DSP) (n = 4 per group).

Article Snippet: AGR2 antibodies and picropodophyllin (PPP, Cat# S7668, Selleck, M€unchen, Germany) were added to the co-culture system.

Techniques: Immunohistochemical staining, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, CRISPR, Knock-Out, Control, Derivative Assay

Journal: Cancers

Article Title: ZEB1/miR-200c/AGR2: A New Regulatory Loop Modulating the Epithelial-Mesenchymal Transition in Lung Adenocarcinomas

doi: 10.3390/cancers12061614

Figure Lengend Snippet: AGR2 negatively correlates with ZEB1 in human cancer cell lines and clinical samples. Determination of AGR2 mRNA expression with respect to ( A ) CDH1 and ( B ) ZEB1 mRNA levels extracted from CBioPortal database containing the Cancer Cell Line Encyclopedia. The value of Spearman’s and Pearson’s correlation coefficient was generated by the CBioPortal database using the default set-up. Scatter plots showed a positive correlation with epithelial CDH1 mRNA (Spearman r = 0.46; Pearson r = 0.47) and a negative correlation between AGR2 and ZEB1 mRNA levels (Spearman r = −0.43; Pearson r = 0.46) in cancer cell lines.

Article Snippet: Cell lysates containing 200 μg of whole proteins were incubated with 1 μg/mL anti-AGR2 mouse antibody (Abnova, Heidelberg, Germany) overnight at 4 °C with gentle agitation.

Techniques: Expressing, Generated

Journal: Cancers

Article Title: ZEB1/miR-200c/AGR2: A New Regulatory Loop Modulating the Epithelial-Mesenchymal Transition in Lung Adenocarcinomas

doi: 10.3390/cancers12061614

Figure Lengend Snippet: ZEB1 regulates AGR2 at mRNA and protein level. ( A ) The expression of ZEB1 was silenced with a specific siRNA (siZEB1) and the changes in AGR2 protein level were determined by Western blot analysis. β-actin served as a loading control. ( B ) Quantitative analysis of AGR2 mRNA was performed in A549 and H1299 cells transfected with specific siRNAs against ZEB1 or control siRNA (siCTR) . 18S rRNA (ribosomal RNA) served as an endogenous control for data normalization. ( C ) Western blot analysis in A549 cells transfected with plasmid coding for ZEB1 or control plasmid (CTR). ( D ) A549 and H1299 cells were transfected with plasmid coding for ZEB1 to determine the effect of enhanced ZEB1 expression on AGR2 mRNA levels by Real-Time PCR (polymerase chain reaction). 18S rRNA served as an endogenous control for data normalization. Data plotted into the graphs are the mean ± standard deviation (SD) obtained from three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. Full Western Blots are shown on .

Article Snippet: Cell lysates containing 200 μg of whole proteins were incubated with 1 μg/mL anti-AGR2 mouse antibody (Abnova, Heidelberg, Germany) overnight at 4 °C with gentle agitation.

Techniques: Expressing, Western Blot, Control, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Standard Deviation

Journal: Cancers

Article Title: ZEB1/miR-200c/AGR2: A New Regulatory Loop Modulating the Epithelial-Mesenchymal Transition in Lung Adenocarcinomas

doi: 10.3390/cancers12061614

Figure Lengend Snippet: ZEB1 directly binds to the promotor of the AGR2 gene to repress its expression under basal conditions. ( A ) Schematic representation of potential E-boxes located in the AGR2 promoter. Blue arrows indicate primer binding sites used for the chromatin immunoprecipitation (ChIP) assay. ( B ) ZEB1 associates with the AGR2 promoter in A549 cells as shown by ChIP analysis. Input DNA samples were used for normalization and rabbit ZEB1 antibody or control immunoglobulins (IgG) were used for ChIP experiments. * p ≤ 0.05. ( C ) AGR2 negative cells H1299 were transfected with specific siRNA against ZEB1 (siZEB1) or control siRNA (siCTR) for 24 hours and with plasmid coding for AGR2 promoter sequence from −1584 to +94 or with control pGL3-luc plasmid for additional 24 hours and the luciferase signal was determined by a spectrophotometer, whereas a Renilla luciferase empty plasmid was used for normalization of the transfection efficiency. Data plotted into the graphs are the mean ± standard deviation (SD) obtained from three independent experiments. ** p ≤ 0.01.

Article Snippet: Cell lysates containing 200 μg of whole proteins were incubated with 1 μg/mL anti-AGR2 mouse antibody (Abnova, Heidelberg, Germany) overnight at 4 °C with gentle agitation.

Techniques: Expressing, Binding Assay, Chromatin Immunoprecipitation, Control, Transfection, Plasmid Preparation, Sequencing, Luciferase, Spectrophotometry, Standard Deviation

Journal: Cancers

Article Title: ZEB1/miR-200c/AGR2: A New Regulatory Loop Modulating the Epithelial-Mesenchymal Transition in Lung Adenocarcinomas

doi: 10.3390/cancers12061614

Figure Lengend Snippet: The knockout of AGR2 alters the mRNA expression of ZEB1. Comparison of ZEB1 mRNA (left) and ZEB1 protein levels (right) in ( A ) A549 scr and A549 KOAGR2 cells, ( B ) H1299 and H1299 AGR2 cells, ( C ) HEK-293 CTR and HEK-293 AGR2 cells, ( D ) A431 CTR and A431 AGR2 cells. The data were normalized using 18S rRNA for mRNA and β-actin for protein expression. The mRNA levels of ZEB1 were determined relative to its mRNA level in A549 scr cells. The results represent the mean ± SD of at least two independent experiments ( A , B ) and one experiment ( C , D ), respectively performed in technical triplicates; ** p ≤ 0.01, *** p ≤ 0.001. ( E ) Cells were transfected with Renilla luciferase reporter plasmid pLuc-CDS coding full-length ZEB1 to analyze the relative amount of ZEB1 in A549 scr and KOAGR2 cells either treated or untreated with TGF-β. ( F ) Immunochemical analysis of ZEB1 protein levels in cells either untreated or treated with TGF-β. ( G ) The subcellular fractionation and immunochemical analysis of ZEB1 protein in nuclear and cytoplasmic fraction. α-tubulin was used as a marker of cytoplasmic fraction and Lamin B1 was used as a marker for nuclear fraction. Full Western Blots of ( A , B ) are shown on . Full Western Blots of ( F , G ) are shown on .

Article Snippet: Cell lysates containing 200 μg of whole proteins were incubated with 1 μg/mL anti-AGR2 mouse antibody (Abnova, Heidelberg, Germany) overnight at 4 °C with gentle agitation.

Techniques: Knock-Out, Expressing, Comparison, Transfection, Luciferase, Plasmid Preparation, Fractionation, Marker, Western Blot

Journal: Cancers

Article Title: ZEB1/miR-200c/AGR2: A New Regulatory Loop Modulating the Epithelial-Mesenchymal Transition in Lung Adenocarcinomas

doi: 10.3390/cancers12061614

Figure Lengend Snippet: AGR2 regulates the stability of the ZEB1 mRNA level. Analysis of the ZEB1 mRNA level in ( A ) A549 scr and A549 KOAGR2 cells and ( B ) H1299 and H1299 AGR2 cells exposed to actinomycin D as indicated. ( C ) RT-qPCR analysis of the miR-200c level in A549 scr and A549 KOAGR2 cells. RNU48 was used as housekeeping control for microRNA (miRNA) levels. ** p ≤ 0.01 ( D ) Protein-protein immunoprecipitation with a specific antibody recognizing AGR2, followed by immunochemical analysis of hnRNPU protein in A549 cells. A non-specific antibody was used as a negative control (NC) for the immunoprecipitation experiments. Full Western Blots are shown on .

Article Snippet: Cell lysates containing 200 μg of whole proteins were incubated with 1 μg/mL anti-AGR2 mouse antibody (Abnova, Heidelberg, Germany) overnight at 4 °C with gentle agitation.

Techniques: Quantitative RT-PCR, Control, Immunoprecipitation, Negative Control, Western Blot

Journal: Cancers

Article Title: ZEB1/miR-200c/AGR2: A New Regulatory Loop Modulating the Epithelial-Mesenchymal Transition in Lung Adenocarcinomas

doi: 10.3390/cancers12061614

Figure Lengend Snippet: The alteration in AGR2 expression regulates an aggressive phenotype in vitro and in vivo. ( A ) Illustration of primary tumors developed in mouse xenografts. A549 scr ( n = 5) and A549 KOAGR2 ( n = 6) cells were injected into the flanks of mice with severe combined immunodeficiency (SCID). ( B ) Growth rate of tumors developed from A549 scr and A549 KOAGR2 cells. ( C ) The graph showing the frequency of metastasis development derived from . ( D ) An invasion assay was performed to analyze the number of cells that invaded through a semi permeable membrane. Invaded cells were stained and measured by spectrophotometer. The results of invasion experiments are an average of 3 technical replicates from 2 independent experiments, plotted as a mean ± SD where ** is ** p ≤ 0.01 and “ns” means statistically non-significant ( p > 0.05).

Article Snippet: Cell lysates containing 200 μg of whole proteins were incubated with 1 μg/mL anti-AGR2 mouse antibody (Abnova, Heidelberg, Germany) overnight at 4 °C with gentle agitation.

Techniques: Expressing, In Vitro, In Vivo, Injection, Derivative Assay, Invasion Assay, Membrane, Staining, Spectrophotometry

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

doi: 10.1038/labinvest.2014.138

Figure Lengend Snippet: Figure 1 Immunohistochemical analysis of anterior gradient 2 (AGR2) in chronic pancreatitis and pre-cancerous lesions. AGR2 expression was not detected in non-neoplastic ducts of chronic pancreatitis patients (a). AGR2 upregulation was observed in benign PDAC precursor lesions including PanIN1A (b) and IPMN with low-grade dysplasia (e), and expression was retained in more severe precursor lesions (c, d and f). PanIN, pancreatic intraepithelial neoplasia; IPMN-L, intraductal papillary mucinous neoplasm with low-grade dysplasia; IPMN-I, intraductal papillary mucinous neoplasm with intermediate-grade dysplasia; (original magnification: (a) 100, (b) 200, (c) 200, (d) 40, (e) 100, (f) 100).

Article Snippet: Primary antibodies used for immunohistochemical (IHC) analysis were as follows: AGR2 (rabbit polyclonal, IMG-5279A, 1/100 dilution, IMGENEX, San Diego, CA, USA), vimentin (mouse monoclonal, Clone V9, 1/25 dilution, DAKO Cytomation, Glostrup, Denmark) and E-cadherin (mouse monoclonal, 610181, 1/1000 dilution, BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Immunohistochemical staining, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

doi: 10.1038/labinvest.2014.138

Figure Lengend Snippet: Figure 2 Histological analyses of human PDAC specimens. Representative images of hematoxylin and eosin (H&E) staining (a–d) and immunohistochemical staining for anterior gradient 2 (AGR2) (e–h), E-cadherin (i–l), and vimentin (m–p). Each column of figures represent serial sections of one PDAC patient (a, e, i and m: well differentiated; b, f, j and n: moderately differentiated; c, g, k and o: poorly differentiated; d, h, l and p: admixed components of well- (arrowhead) and poorly (asterisk) differentiated adenocarcinoma. Low-histological-cellular-grade (well or moderately differentiated) PDAC retained AGR2 and membranous E-cadherin expression, whereas vimentin expression was absent. Cytoplasmic AGR2 expression and membranous E-cadherin expression were decreased in parallel with cancer de-differentiation, and positive cytoplasmic vimentin expression was observed in high-histological-cellular-grade (poorly differentiated) PDAC. E-cadherin expression is seen at the membrane and in the cytoplasm of the well-differentiated carcinoma, but is decreased in the membrane of the poorly differentiated carcinoma (l; insets). Original magnification: H&E staining 100, IHC 200.

Article Snippet: Primary antibodies used for immunohistochemical (IHC) analysis were as follows: AGR2 (rabbit polyclonal, IMG-5279A, 1/100 dilution, IMGENEX, San Diego, CA, USA), vimentin (mouse monoclonal, Clone V9, 1/25 dilution, DAKO Cytomation, Glostrup, Denmark) and E-cadherin (mouse monoclonal, 610181, 1/1000 dilution, BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Staining, Immunohistochemical staining, Expressing, Membrane

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

doi: 10.1038/labinvest.2014.138

Figure Lengend Snippet: Figure 3 Kaplan–Meier survival curves demonstrating disease-free survival in PDAC patients according to anterior gradient 2 (AGR2) and E-cadherin expression. Low AGR2 expression was associated with poor prognosis in disease-free survival (a; log-rank test; Po0.0001). Disease-free survival rate with combined AGR2 and E-cadherin status (b). In both high- and low-E-cadherin expression cohorts, low AGR2 expression was associated with a lower survival rate (c and d).

Article Snippet: Primary antibodies used for immunohistochemical (IHC) analysis were as follows: AGR2 (rabbit polyclonal, IMG-5279A, 1/100 dilution, IMGENEX, San Diego, CA, USA), vimentin (mouse monoclonal, Clone V9, 1/25 dilution, DAKO Cytomation, Glostrup, Denmark) and E-cadherin (mouse monoclonal, 610181, 1/1000 dilution, BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

doi: 10.1038/labinvest.2014.138

Figure Lengend Snippet: Figure 4 Quantitative reverse transcription-PCR for anterior gradient 2 (AGR2) expression in pancreatic cancer cell lines. AGR2 expression differed in the various pancreatic cancer cell lines. High AGR2-expressing cell lines exhibited overexpression of vimentin and reduced E-cadherin expression. Epithelial-type cancer cells (HS766T, H48N, and BxPC-3), retained cellular adhesiveness and AGR2 expression, whereas mesenchymal-type cancer cell lines (MiaPaCa2, SUIT-2, and HPC-3) exhibiting spindle-shaped morphology expressed low levels of AGR2. AGR2 and E-cadherin expression was very low and vimentin expression was quite high in the primary-cultured pancreatic stellate cells (PSCs) (a). Linear regression analysis demonstrated a strong positive correlation between AGR2 and E-cadherin messenger RNA (mRNA) levels (b: R2 ¼ 0.7262, P ¼ 0.0035).

Article Snippet: Primary antibodies used for immunohistochemical (IHC) analysis were as follows: AGR2 (rabbit polyclonal, IMG-5279A, 1/100 dilution, IMGENEX, San Diego, CA, USA), vimentin (mouse monoclonal, Clone V9, 1/25 dilution, DAKO Cytomation, Glostrup, Denmark) and E-cadherin (mouse monoclonal, 610181, 1/1000 dilution, BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Reverse Transcription, Expressing, Over Expression, Cell Culture

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

doi: 10.1038/labinvest.2014.138

Figure Lengend Snippet: Figure 5 Targeted inhibition of anterior gradient 2 (AGR2) using small-interfering RNA (siRNA). AGR2 silencing was confirmed following transfection of Aspc-1 and Hs766T cells with AGR2-specific siRNA by quantitative reverse transcription (qRT)-PCR (a) 48, 72, and 120 h, and immunoblot analysis 72 h post transfection, respectively (b). E-cadherin and vimentin expression levels were not altered by AGR2 knockdown as assessed by immunoblot analysis (b). AGR2 knockdown significantly reduced cell proliferation (c), colony formation (d), cell migration (e), and cell invasiveness (f) in Aspc-1 and Hs766T cells (Po0.01), except for the colony formation assay in Hs766T cells (P ¼ 0.0686). AGR2 knockdown did not alter messenger RNA (mRNA) levels of epithelial–mesenchymal transition markers, E-cadherin, vimentin, Snail-1, Snail-2, and ZEB-1 (g). Data represent the mean values of triplicate experiments (*Po0.01, versus control). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Primary antibodies used for immunohistochemical (IHC) analysis were as follows: AGR2 (rabbit polyclonal, IMG-5279A, 1/100 dilution, IMGENEX, San Diego, CA, USA), vimentin (mouse monoclonal, Clone V9, 1/25 dilution, DAKO Cytomation, Glostrup, Denmark) and E-cadherin (mouse monoclonal, 610181, 1/1000 dilution, BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Inhibition, Small Interfering RNA, Transfection, Reverse Transcription, Quantitative RT-PCR, Western Blot, Expressing, Knockdown, Migration, Colony Assay, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

doi: 10.1038/labinvest.2014.138

Figure Lengend Snippet: Figure 6 Indirect co-culture experiment of pancreatic ductal adenocarcinoma (PDAC) cells with pancreatic stellate cell (PSCs) and treatment with recombinant growth factors. Anterior gradient 2 (AGR2) expression was decreased in PDAC cells co-cultured with PSCs using an indirect co-culture system (a) in accordance with epithelial–mesenchymal transition induction (E-cadherin downregulation (b) and overexpression of vimentin, Snail-1, Snail-2 and ZEB-1 (c)) by quantitative reverse transcription-PCR (*Po0.01, versus monocultures). Decreased expression of AGR2 was observed in co- cultured cancer cells compared with monocultures. Decreased AGR2 expression was also associated with increased levels of phosphorylated Smad2/3 and ERK1/2, with the exception of phosphorylated Smad2/3 in Aspc-1 (d). E-cadherin and vimentin expression were also slightly altered at the protein level (d). Enzyme-linked immunosorbent assays revealed that total, secreted transforming growth factor beta-1 (TGF-b1) levels were higher in co-culture supernatants than that of monocultures (e: *Po0.001, versus the medium of Aspc-1 monocultures). Treatment with recombinant proteins showed that reduced expression of AGR2 was observed by TGF-b1 in all PDAC cells examined and by high-dose epidermal growth factor (EGF) in Aspc-1 and Capan-2 cells (f). AGR2 expression was decreased by TGF-b1 or co-cultured with PSCs, and restored by inhibition of TGF-b signaling (g). Data represent the mean values of triplicate experiments (*Po0.01, versus control). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; FGF-2, fibroblast growth factor 2; mRNA, messenger RNA.

Article Snippet: Primary antibodies used for immunohistochemical (IHC) analysis were as follows: AGR2 (rabbit polyclonal, IMG-5279A, 1/100 dilution, IMGENEX, San Diego, CA, USA), vimentin (mouse monoclonal, Clone V9, 1/25 dilution, DAKO Cytomation, Glostrup, Denmark) and E-cadherin (mouse monoclonal, 610181, 1/1000 dilution, BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Co-Culture Assay, Recombinant, Expressing, Cell Culture, Over Expression, Reverse Transcription, Inhibition, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

doi: 10.1038/labinvest.2014.138

Figure Lengend Snippet: Figure 7 Schematic summarising the mechanisms of anterior gradient 2 (AGR2) downregulation. AGR2 expression was not observed in non-neoplastic epithelium of normal pancreas or chronic pancreatitis, but was upregulated in intraepithelial neoplastic lesions and contributed to cancer development. As cancer cells invade the stroma through the basement membrane, EMT is induced following interaction with stromal cells, including PSCs. AGR2 is downregulated by TGF-b1 secreted from PSCs. Cancer progression is slightly reduced by AGR2 downregulation; however, the various oncogenic factors, upregulated in advanced PDAC, retain or promote cancer progression. (Nor/CP, pancreatic ductal epithelium of normal pancreas and chronic pancreatitis; PanIN, pancreatic intraepithelial epithelium; PDAC, pancreatic ductal adenocarcinoma; EMT, epithelial–mesenchymal transition; PSCs, pancreatic stellate cells; TGF-b1, transforming growth factor beta-1).

Article Snippet: Primary antibodies used for immunohistochemical (IHC) analysis were as follows: AGR2 (rabbit polyclonal, IMG-5279A, 1/100 dilution, IMGENEX, San Diego, CA, USA), vimentin (mouse monoclonal, Clone V9, 1/25 dilution, DAKO Cytomation, Glostrup, Denmark) and E-cadherin (mouse monoclonal, 610181, 1/1000 dilution, BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Expressing, Membrane

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

doi: 10.1038/labinvest.2014.138

Figure Lengend Snippet: Figure 1 Immunohistochemical analysis of anterior gradient 2 (AGR2) in chronic pancreatitis and pre-cancerous lesions. AGR2 expression was not detected in non-neoplastic ducts of chronic pancreatitis patients (a). AGR2 upregulation was observed in benign PDAC precursor lesions including PanIN1A (b) and IPMN with low-grade dysplasia (e), and expression was retained in more severe precursor lesions (c, d and f). PanIN, pancreatic intraepithelial neoplasia; IPMN-L, intraductal papillary mucinous neoplasm with low-grade dysplasia; IPMN-I, intraductal papillary mucinous neoplasm with intermediate-grade dysplasia; (original magnification: (a) 100, (b) 200, (c) 200, (d) 40, (e) 100, (f) 100).

Article Snippet: Membranes were blocked with 5% dry skimmed milk and incubated with primary antibodies; anti-AGR2 rabbit polyclonal antibody (1:500, IMGENEX), anti-E-cadherin mouse polyclonal antibody (1:5000, BD Biosciences), anti-vimentin mouse monoclonal antibody (1:500, DAKO Cytomation), anti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) rabbit monoclonal antibody (#8828, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-Smad2/3 rabbit monoclonal antibody (#8685, 1:1000, Cell Signaling Technology), antiphospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit monoclonal antibody (#4370, 1:2000, Cell Signaling Technology), and anti-p44/42 MAPK (Erk1/2) rabbit monoclonal antibody (#4695, 1:1000, Cell Signaling Technology) using the SNAP i.d.

Techniques: Immunohistochemical staining, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

doi: 10.1038/labinvest.2014.138

Figure Lengend Snippet: Figure 2 Histological analyses of human PDAC specimens. Representative images of hematoxylin and eosin (H&E) staining (a–d) and immunohistochemical staining for anterior gradient 2 (AGR2) (e–h), E-cadherin (i–l), and vimentin (m–p). Each column of figures represent serial sections of one PDAC patient (a, e, i and m: well differentiated; b, f, j and n: moderately differentiated; c, g, k and o: poorly differentiated; d, h, l and p: admixed components of well- (arrowhead) and poorly (asterisk) differentiated adenocarcinoma. Low-histological-cellular-grade (well or moderately differentiated) PDAC retained AGR2 and membranous E-cadherin expression, whereas vimentin expression was absent. Cytoplasmic AGR2 expression and membranous E-cadherin expression were decreased in parallel with cancer de-differentiation, and positive cytoplasmic vimentin expression was observed in high-histological-cellular-grade (poorly differentiated) PDAC. E-cadherin expression is seen at the membrane and in the cytoplasm of the well-differentiated carcinoma, but is decreased in the membrane of the poorly differentiated carcinoma (l; insets). Original magnification: H&E staining 100, IHC 200.

Article Snippet: Membranes were blocked with 5% dry skimmed milk and incubated with primary antibodies; anti-AGR2 rabbit polyclonal antibody (1:500, IMGENEX), anti-E-cadherin mouse polyclonal antibody (1:5000, BD Biosciences), anti-vimentin mouse monoclonal antibody (1:500, DAKO Cytomation), anti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) rabbit monoclonal antibody (#8828, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-Smad2/3 rabbit monoclonal antibody (#8685, 1:1000, Cell Signaling Technology), antiphospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit monoclonal antibody (#4370, 1:2000, Cell Signaling Technology), and anti-p44/42 MAPK (Erk1/2) rabbit monoclonal antibody (#4695, 1:1000, Cell Signaling Technology) using the SNAP i.d.

Techniques: Staining, Immunohistochemical staining, Expressing, Membrane

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

doi: 10.1038/labinvest.2014.138

Figure Lengend Snippet: Figure 3 Kaplan–Meier survival curves demonstrating disease-free survival in PDAC patients according to anterior gradient 2 (AGR2) and E-cadherin expression. Low AGR2 expression was associated with poor prognosis in disease-free survival (a; log-rank test; Po0.0001). Disease-free survival rate with combined AGR2 and E-cadherin status (b). In both high- and low-E-cadherin expression cohorts, low AGR2 expression was associated with a lower survival rate (c and d).

Article Snippet: Membranes were blocked with 5% dry skimmed milk and incubated with primary antibodies; anti-AGR2 rabbit polyclonal antibody (1:500, IMGENEX), anti-E-cadherin mouse polyclonal antibody (1:5000, BD Biosciences), anti-vimentin mouse monoclonal antibody (1:500, DAKO Cytomation), anti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) rabbit monoclonal antibody (#8828, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-Smad2/3 rabbit monoclonal antibody (#8685, 1:1000, Cell Signaling Technology), antiphospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit monoclonal antibody (#4370, 1:2000, Cell Signaling Technology), and anti-p44/42 MAPK (Erk1/2) rabbit monoclonal antibody (#4695, 1:1000, Cell Signaling Technology) using the SNAP i.d.

Techniques: Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

doi: 10.1038/labinvest.2014.138

Figure Lengend Snippet: Figure 4 Quantitative reverse transcription-PCR for anterior gradient 2 (AGR2) expression in pancreatic cancer cell lines. AGR2 expression differed in the various pancreatic cancer cell lines. High AGR2-expressing cell lines exhibited overexpression of vimentin and reduced E-cadherin expression. Epithelial-type cancer cells (HS766T, H48N, and BxPC-3), retained cellular adhesiveness and AGR2 expression, whereas mesenchymal-type cancer cell lines (MiaPaCa2, SUIT-2, and HPC-3) exhibiting spindle-shaped morphology expressed low levels of AGR2. AGR2 and E-cadherin expression was very low and vimentin expression was quite high in the primary-cultured pancreatic stellate cells (PSCs) (a). Linear regression analysis demonstrated a strong positive correlation between AGR2 and E-cadherin messenger RNA (mRNA) levels (b: R2 ¼ 0.7262, P ¼ 0.0035).

Article Snippet: Membranes were blocked with 5% dry skimmed milk and incubated with primary antibodies; anti-AGR2 rabbit polyclonal antibody (1:500, IMGENEX), anti-E-cadherin mouse polyclonal antibody (1:5000, BD Biosciences), anti-vimentin mouse monoclonal antibody (1:500, DAKO Cytomation), anti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) rabbit monoclonal antibody (#8828, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-Smad2/3 rabbit monoclonal antibody (#8685, 1:1000, Cell Signaling Technology), antiphospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit monoclonal antibody (#4370, 1:2000, Cell Signaling Technology), and anti-p44/42 MAPK (Erk1/2) rabbit monoclonal antibody (#4695, 1:1000, Cell Signaling Technology) using the SNAP i.d.

Techniques: Reverse Transcription, Expressing, Over Expression, Cell Culture

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

doi: 10.1038/labinvest.2014.138

Figure Lengend Snippet: Figure 5 Targeted inhibition of anterior gradient 2 (AGR2) using small-interfering RNA (siRNA). AGR2 silencing was confirmed following transfection of Aspc-1 and Hs766T cells with AGR2-specific siRNA by quantitative reverse transcription (qRT)-PCR (a) 48, 72, and 120 h, and immunoblot analysis 72 h post transfection, respectively (b). E-cadherin and vimentin expression levels were not altered by AGR2 knockdown as assessed by immunoblot analysis (b). AGR2 knockdown significantly reduced cell proliferation (c), colony formation (d), cell migration (e), and cell invasiveness (f) in Aspc-1 and Hs766T cells (Po0.01), except for the colony formation assay in Hs766T cells (P ¼ 0.0686). AGR2 knockdown did not alter messenger RNA (mRNA) levels of epithelial–mesenchymal transition markers, E-cadherin, vimentin, Snail-1, Snail-2, and ZEB-1 (g). Data represent the mean values of triplicate experiments (*Po0.01, versus control). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Membranes were blocked with 5% dry skimmed milk and incubated with primary antibodies; anti-AGR2 rabbit polyclonal antibody (1:500, IMGENEX), anti-E-cadherin mouse polyclonal antibody (1:5000, BD Biosciences), anti-vimentin mouse monoclonal antibody (1:500, DAKO Cytomation), anti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) rabbit monoclonal antibody (#8828, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-Smad2/3 rabbit monoclonal antibody (#8685, 1:1000, Cell Signaling Technology), antiphospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit monoclonal antibody (#4370, 1:2000, Cell Signaling Technology), and anti-p44/42 MAPK (Erk1/2) rabbit monoclonal antibody (#4695, 1:1000, Cell Signaling Technology) using the SNAP i.d.

Techniques: Inhibition, Small Interfering RNA, Transfection, Reverse Transcription, Quantitative RT-PCR, Western Blot, Expressing, Knockdown, Migration, Colony Assay, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

doi: 10.1038/labinvest.2014.138

Figure Lengend Snippet: Figure 6 Indirect co-culture experiment of pancreatic ductal adenocarcinoma (PDAC) cells with pancreatic stellate cell (PSCs) and treatment with recombinant growth factors. Anterior gradient 2 (AGR2) expression was decreased in PDAC cells co-cultured with PSCs using an indirect co-culture system (a) in accordance with epithelial–mesenchymal transition induction (E-cadherin downregulation (b) and overexpression of vimentin, Snail-1, Snail-2 and ZEB-1 (c)) by quantitative reverse transcription-PCR (*Po0.01, versus monocultures). Decreased expression of AGR2 was observed in co- cultured cancer cells compared with monocultures. Decreased AGR2 expression was also associated with increased levels of phosphorylated Smad2/3 and ERK1/2, with the exception of phosphorylated Smad2/3 in Aspc-1 (d). E-cadherin and vimentin expression were also slightly altered at the protein level (d). Enzyme-linked immunosorbent assays revealed that total, secreted transforming growth factor beta-1 (TGF-b1) levels were higher in co-culture supernatants than that of monocultures (e: *Po0.001, versus the medium of Aspc-1 monocultures). Treatment with recombinant proteins showed that reduced expression of AGR2 was observed by TGF-b1 in all PDAC cells examined and by high-dose epidermal growth factor (EGF) in Aspc-1 and Capan-2 cells (f). AGR2 expression was decreased by TGF-b1 or co-cultured with PSCs, and restored by inhibition of TGF-b signaling (g). Data represent the mean values of triplicate experiments (*Po0.01, versus control). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; FGF-2, fibroblast growth factor 2; mRNA, messenger RNA.

Article Snippet: Membranes were blocked with 5% dry skimmed milk and incubated with primary antibodies; anti-AGR2 rabbit polyclonal antibody (1:500, IMGENEX), anti-E-cadherin mouse polyclonal antibody (1:5000, BD Biosciences), anti-vimentin mouse monoclonal antibody (1:500, DAKO Cytomation), anti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) rabbit monoclonal antibody (#8828, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-Smad2/3 rabbit monoclonal antibody (#8685, 1:1000, Cell Signaling Technology), antiphospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit monoclonal antibody (#4370, 1:2000, Cell Signaling Technology), and anti-p44/42 MAPK (Erk1/2) rabbit monoclonal antibody (#4695, 1:1000, Cell Signaling Technology) using the SNAP i.d.

Techniques: Co-Culture Assay, Recombinant, Expressing, Cell Culture, Over Expression, Reverse Transcription, Inhibition, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Anterior gradient 2 downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.

doi: 10.1038/labinvest.2014.138

Figure Lengend Snippet: Figure 7 Schematic summarising the mechanisms of anterior gradient 2 (AGR2) downregulation. AGR2 expression was not observed in non-neoplastic epithelium of normal pancreas or chronic pancreatitis, but was upregulated in intraepithelial neoplastic lesions and contributed to cancer development. As cancer cells invade the stroma through the basement membrane, EMT is induced following interaction with stromal cells, including PSCs. AGR2 is downregulated by TGF-b1 secreted from PSCs. Cancer progression is slightly reduced by AGR2 downregulation; however, the various oncogenic factors, upregulated in advanced PDAC, retain or promote cancer progression. (Nor/CP, pancreatic ductal epithelium of normal pancreas and chronic pancreatitis; PanIN, pancreatic intraepithelial epithelium; PDAC, pancreatic ductal adenocarcinoma; EMT, epithelial–mesenchymal transition; PSCs, pancreatic stellate cells; TGF-b1, transforming growth factor beta-1).

Article Snippet: Membranes were blocked with 5% dry skimmed milk and incubated with primary antibodies; anti-AGR2 rabbit polyclonal antibody (1:500, IMGENEX), anti-E-cadherin mouse polyclonal antibody (1:5000, BD Biosciences), anti-vimentin mouse monoclonal antibody (1:500, DAKO Cytomation), anti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) rabbit monoclonal antibody (#8828, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-Smad2/3 rabbit monoclonal antibody (#8685, 1:1000, Cell Signaling Technology), antiphospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit monoclonal antibody (#4370, 1:2000, Cell Signaling Technology), and anti-p44/42 MAPK (Erk1/2) rabbit monoclonal antibody (#4695, 1:1000, Cell Signaling Technology) using the SNAP i.d.

Techniques: Expressing, Membrane

Journal: eLife

Article Title: Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties

doi: 10.7554/eLife.13887

Figure Lengend Snippet: Confocal cross-sections of HBEC organoids in the presence (+eAGR2) or absence (-eAGR2) of AGR2 in the ECM stained with E-Cadherin ( A ), β-catenin ( B ), or Laminin-V ( C ), and DAPI (blue) for nucleus. Scale bars, 50 μm. DOI: http://dx.doi.org/10.7554/eLife.13887.014

Article Snippet: The sources of the primary antibodies used in these studies were as follows: β-catenin (BD Biosciences), cleaved-caspase 3 (Asp175) (Cell Signaling), Calnexin (Stressgen), GM130 (BD Transduction Laboratories), laminin V (Millipore), mouse AGR2 (Abnova, clone 1C3), rabbit polyclonal AGR2 antibody (Abcam), BiP antibody (Abcam), GAPDH antibody (Millipore), Transferrin antibody (Cell signalling), and O-linked-N-acetylglucosamine antibody (Abcam).

Techniques: Staining