Journal: Oncology reports

Article Title: Establishment and characterization of a novel human cholangiocarcinoma cell line with high metastatic activity.

doi: 10.3892/or.2016.4974

Figure Lengend Snippet: Figure 6. AGR2 and KiSS-1 expression in CCA patient tissues. Immunohistochemistry of AGR2 and KiSS-1 was performed in 32 cases of intrahepatic mass-forming CCA patient tissues with different tumor staging (A). The immunohistochemical (IHC) indices were determined in normal bile duct (NBD, n=30), CCA stage III (n=10), CCA stage IVA (n=11) and CCA stage IVB (n=11). Normal bile duct epithelia from adjacent non-tumorous tissues were assessed. Semi-quantitative analysis of AGR2 and KiSS-1 expression levels presented as IHC indices are shown in (B); *P<0.05, ***P<0.001.

Article Snippet: AGR2 and KiSS-1 immunohistochemistry was performed in 32 intrahepatic mass-forming CCA patient tissues using 1:500 rabbit polyclonal anti-AGR2 antibody (Abcam, Cambridge, MA, USA) and 1:50 rabbit polyclonal anti-KiSS-1 antibody (Santa Cruz biotechnology, Dallas, TX, USA) at room temperature, overnight.

Techniques: Expressing, Immunohistochemistry, Immunohistochemical staining

Journal: Nature

Article Title: A gene-environment induced epigenetic program initiates tumorigenesis

doi: 10.1038/s41586-020-03147-x

Figure Lengend Snippet: a, Schematic representation of the experimental design to interrogate the impact of recombinant IL-33 (rIL-33) on the transcriptional, chromatin and phenotypic state of the pancreatic epithelium from Kras-mutant (KC-GEMM) or wild-type (C-GEMM) mice. Molecular analyses were performed in lineage-traced (mKate2+) pancreatic epithelial cells purified by FACS-sorting from of rIL-33 or vehicle treated mice at day 0 (ATAC-seq), or day 0 and day 21 days (RNA-seq) after treatment. b, GSEA comparing the expression of the early chromatin activated gene program identified in analyses (left), or of genes overexpressed in human PDAC specimens compared to normal pancreas (Moffitt et al. dataset) (right), in Kras-mutant cells isolated from rIL-33 treated vs PBS-treated mice (day 21 time point). The chromatin activated genes queried are the chromatin-dynamic DEGs identified to be upregulated during injury-accelerated neoplasia ( Kras*+Injury ) and in advanced disease ( PDAC ) but not during normal regeneration ( Injury alone) and blunted by Brd4 suppression in metaplastic Kras-mutant cells (KC sh : Kras+Injury ). c-d, GSEA comparing the expression of genes induced by the combination of mutant Kras + rIL-33 in either shBrd4 vs shRen Kras-mutant pancreatic epithelial cells (mKate2+) isolated from KC sh -GEMM ( Kras*+Injury ) (c) or in Kras-mutant populations isolated from caeruelin-treated ( Kras*+Injury ) vs resting ( Kras* ) KC mice (d). The queried gene sets were identified as significantly upregulated in Kras-mutant pancreatic epithelial cell populations (mKate2+) isolated from rIL-33 (vs PBS) treated mice ( KC+rIL-33 vs KC+Veh ) at either day 0 (d0) or day 21 (d21) time points. e, qRT-PCR analysis of rIL-33 effects in the mRNA levels of acinar differentiation ( Cpa1 ), metaplasia ( Sox9 ) and Kras-dependent neoplasia ( Agr2 , Muc6 ) markers in pancreatic epithelial cell (mKate2+) populations isolated from Kras-WT (C) or Kras-mutant (KC) mice (n=2 each) treated with rIL-33 or Vehicle (PBS) and analyzed 21 days thereafter. f, GSEA comparing the expression of genes induced by the combination of mutant Kras + rIL-33 in human PDAC specimens vs human normal pancreas (Moffitt et al. dataset) . g, Volcano plots comparing the chromatin accessibility landscape of Kras-mutant pancreatic epithelium of rIL-33-treated vs vehicle-treated mice, as assessed by ATAC-seq performed at the day 0 time-point. h, Top-scoring motifs identified by HOMER de novo analysis in accessibility- GAIN peaks identified in Kras-mutant pancreatic epithelial cells (mKate2+) isolated from rIL-33-treated mice vs from PBS-treated counterparts, assessed by ATAC-seq analyses performed at the day 0 time point. The significance of the enrichment is shown in brackets. i , Metagene representation of the mean ATAC-seq signal (n=3 mice per condition) at accessibility- GAIN regions driven by injury in the Kras-mutant pancreatic epithelium ( Kras*+Injury vs Kras* ) (top) or at accessibility- GAIN regions linked to the neoplasia-specific gene activation program (identified in analyses, right) in Kras-mutant pancreatic epithelial cells (mKate2+) from isolated from rIL-33 treated vs PBS-treated mice (n=3 each, day 0 time point). rIL-33 treatment promotes accessibility at injury-sensitive sites. p-values were determined by Kolmogorov–Smirnov test. j, Quantification of the relative number of ADM and PanIN lesions in pancreata from Kras wild-type (C-GEMM) or Kras mutant (KC-GEMM) mice treated with rIL-33 or vehicle (PBS) and analyzed at the indicated time points in days (d) after treatment. Data are presented as means ± s.e.m and significance was assessed by unpaired two-tailed Student’s t-test (ns, not significant). n=3, 4, 4, 5, 3 or 4 (from left to right) independent animals per experimental condition. k, Representative immunofluorescence stains of IL-33 protein (green) co-stained with the lineage-tracer marker mKate2 (red) marking pancreatic epithelial cells from mice (n=3 per group) harbouring wild-type (Normal) or mutant Kras in the indicated tissue states. Scale bar, 100 μm. l, Relative mRNA levels (RNA-seq tpm counts) of Il33 (left) or the indicated mutant Kras effector ( Agr2 ), middle) or acinar TF ( Cpa1 , right) in FACS-sorted mKate2+ pancreatic epithelial cell populations isolated from rIL-33-treated or PBS-treated mice harbouring WT or mutant Kras . n=3, 4, 4, 4, 5 or 4 (from left to right) biological replicates (independent mice) per group; median and upper/lower quantile values per group are indicated.

Article Snippet: The following primary antibodies were used: mKate2 (Evrogen, AB233, 1:1000), GFP (ab13970, Abcam, 1:500; and 2956S, Cell Signaling Technology, 1:200), Brd4 (HPA015055, Sigma-Aldrich, 1:100), Myc (ab32072, Abcam, 1:100), CPA1 (AF2765, R&D, 1:400), Clusterin (sc-6419, SCBT, 1:200), SOX9 (AB5535, Millipore, 1:1000), Amylase (sc-31869, SCBT, 1:1000), KRT19 (Troma III, Developmental Studies Hybridoma Bank, 1:500), FOSL1 (sc-376148, SCBT, 1:100), JUNB (sc-8051, SCBT, 1:100), AGR2 (NBP2–27393, Novus Biologicals, 1:200), DCLK1 (ab109029, Abcam, 1:200), Ki67 (BD Biosciences 550609, 1:200) and IL-33 (AF3626, R&D, 1:150).

Techniques: Recombinant, Mutagenesis, Purification, RNA Sequencing Assay, Expressing, Isolation, Quantitative RT-PCR, Activation Assay, Two Tailed Test, Immunofluorescence, Staining, Marker

Journal: Cancer Research

Article Title: The Adenocarcinoma-Associated Antigen, AGR2, Promotes Tumor Growth, Cell Migration, and Cellular Transformation

doi: 10.1158/0008-5472.can-07-2930

Figure Lengend Snippet: Figure 1. Effects of AGR2 suppression in SEG-1 cells. A, AGR2 expression in SEG-1 cells suppressed with shRNAmir. SEG-1 cells were transduced with shRNAmir retroviral constructs (KD1, KD2, KD3) followed by selection with puromycin. SEG-1 control cells were transduced with the retroviral vector alone (Vector). The chart displays the cell lines assayed by real-time PCR for AGR2 mRNA normalized to h-actin mRNA; and compared with SEG-1 control cells. Inset, AGR2 protein expression assayed with protein immunoblotting. All remaining experiments used the SEG-1:KD1 cells. B, anchorage-independent growth as measured by colony growth in soft agar at different initial plating densities. SEG-1:KD1 cells showed an 82% reduction in colonies with the plating of 1,000 cells. Columns, mean; bars, 1 SD. C, SEG-1 control cells (x) and SEG-1:KD1 cells were grown as tumor xenografts in nude mice (n). Points, mean from five mice; bars, 1 SD. D, migration assay. SEG-1:KD1 cells were grown in filter chambers bathed in conditioned culture media derived from either SEG-1 control cells (AGR2+) or SEG-1:KD1 cells (AGR2). Columns, mean fold change from three experiments performed in duplicate and normalized to the experiments using AGR2-deficient conditioned media; bars, 1 SE. The number of cells which migrated for the AGR2+ media ranged from 133 to 264 cells.

Article Snippet: Rabbit anti-AGR2 antibodies (Imgenex Corp.) were incubated overnight at room temperature at a dilution of 1:100 in PBS.

Techniques: Expressing, Transduction, Retroviral, Construct, Selection, Control, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Migration, Derivative Assay

Journal: Cancer Research

Article Title: The Adenocarcinoma-Associated Antigen, AGR2, Promotes Tumor Growth, Cell Migration, and Cellular Transformation

doi: 10.1158/0008-5472.can-07-2930

Figure Lengend Snippet: Figure 2. Effects of AGR2 expression in NIH3T3 cells. A, focus-formation assay of AGR2-expressing NIH3T3 cells or control cells transfected with the expression vector alone. Top, images of the control cells (left) and a foci of NIH3T3 AGR2 cells (right). Bottom, chart depicting one of three representative experiments. B, anchorage-independent growth as measured by colony growth in soft agar after different initial plating densities of NIH3T3:AGR2 cells or control NIH3T3 cells. Columns, mean from one of two experiments performed in triplicate; bars, 1 SD. C, representative example of NIH3T3 control cells (right) and NIH3T3:AGR2 cells (left) grown as xenografts in BALB/c nude mice. Implantation site of the cells (dotted lines). D, chart depicting the growth of NIH3T3 control cells (x) and NIH3T3:AGR2 expressing cells (n) grown as tumor xenografts. Points, mean of five mice; bars, 1 SD.

Article Snippet: Rabbit anti-AGR2 antibodies (Imgenex Corp.) were incubated overnight at room temperature at a dilution of 1:100 in PBS.

Techniques: Expressing, Tube Formation Assay, Control, Transfection, Plasmid Preparation

Journal: Cancer Research

Article Title: The Adenocarcinoma-Associated Antigen, AGR2, Promotes Tumor Growth, Cell Migration, and Cellular Transformation

doi: 10.1158/0008-5472.can-07-2930

Figure Lengend Snippet: Figure 4. Relationship of AGR2 expression to other intestinal cell fate determinants. A, real-time PCR of AGR2 and GFI1 mRNA in SEG1:KD1 and SEG-1 control cells. AGR2 and GFI1 mRNA levels were normalized to h-actin and represented as the fold-change to SEG-1 control cells. Columns, mean value of samples measured in triplicate (mean Ct values in SEG-1 control cells for AGR2 = 13.6 and GFI1 = 27). B, schematic of expression patterns for AGR2 in the context of basic helix-loop-helix transcription factors known to influence intestinal cell fate (derived from refs. 30, 33, 34, 38, 39). Whether each secretory lineage is derived from a dedicated progenitor cell remains to be determined. ISC, intestinal stem cell. Gene names used are those approved by the HUGO Gene Nomenclature Committee.

Article Snippet: Rabbit anti-AGR2 antibodies (Imgenex Corp.) were incubated overnight at room temperature at a dilution of 1:100 in PBS.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Derivative Assay

Journal: Cancer Research

Article Title: The Adenocarcinoma-Associated Antigen, AGR2, Promotes Tumor Growth, Cell Migration, and Cellular Transformation

doi: 10.1158/0008-5472.can-07-2930

Figure Lengend Snippet: Figure 3. Immunohistochemistry of mouse small intestine. A, small intestinal section in which the goblet cells are labeled with Alcian blue (black arrows) and AGR2 is labeled with horseradish peroxidase stain (brown). Inset, Paneth cells at the bottom of a crypt that stain intensely for AGR2. Arrows, representative cells that stain for Alcian blue. B and C, immunofluorescence of adjacent serial intestinal sections stained for chromogranin A (CHGA) antibodies (B, green) or anti-AGR2 antibodies (C, green). Nuclei in both sections were stained with 4¶,6-diamidino-2-phenylindole (blue). Cells that label for both chromogranin A and AGR2 (white arrows). D to F, triple labeling of an intestinal crypt for MSI1 (E and F, green), AGR2 (D and E, red), and nuclei (D, E, and F, blue). Arrows, cells labeled for AGR2 and MSI1. G to J, a single intestinal crypt labeled for AGR2 (G–J, red), the proliferation marker Ki-67 (H and I, green), and nuclei (I and J, blue). G, phase contrast image of the crypt with overlying AGR2 immunofluorescence. White arrows, cells that label for the nuclear antigen Ki-67 and AGR2, which labels outside the nucleus. Arrowheads, representative Paneth cells at the bottom of the crypt.

Article Snippet: Rabbit anti-AGR2 antibodies (Imgenex Corp.) were incubated overnight at room temperature at a dilution of 1:100 in PBS.

Techniques: Immunohistochemistry, Labeling, Staining, Immunofluorescence, Marker

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: Molecular & Cellular Proteomics : MCP

Article Title: Characterization of the AGR2 Interactome Uncovers New Players of Protein Disulfide Isomerase Network in Cancer Cells

doi: 10.1016/j.mcpro.2021.100188

Figure Lengend Snippet: Workflow of AGR2 pull-down (PD) methods and protein-level analysis. A, two different cell lines were used for AGR2 PD. Breast cancer cell line T47D, of which endogenously expressed AGR2 protein underwent DSP crosslinking and PD with AGR2-specific E7 peptide and untargeted control peptide F4. Cells without DSP treatment served as a control. Corresponding MS data were quantified using label-free quantification (LFQ). H1299 lung carcinoma cell line was stably transfected with vector carrying coding sequence of AGR2. AGR2 positive clone was labeled with heavy (R10K8) SILAC medium, whereas parental H1299 served as a control and was maintained in light (R0K0) SILAC medium. These cells underwent DSP crosslinking, and lysates were, according to total protein content, equally mixed into one sample, and PD was done with E7 (AGR2 specific) and F4 (control) peptides again. MS data were quantified using SILAC approach. B and C, protein levels of AGR2 were detected in all input samples (20 μg of total protein per well) as well as in eluted proteins (10 μl of eluates per well) using immunoblotting with anti-AGR2 and antiactin antibodies, which served as a loading control. Numbers under the bands represent integral absorbance (INT∗mm 2 ∗10 3 ) obtained by Quantity One software (Bio-Rad). AGR2, anterior gradient 2; DSP, dithiobis[succinimidylpropionate]; MS, mass spectrometry; SILAC, stable isotope labeling with amino acids in cell culture.

Article Snippet: In contrast, cells labeled with two different fluorochromes for AGR2 (obtained by anti-AGR2 mouse Ab from Abnova and anti-AGR2 rabbit Ab from Abcam followed by fluorochrome-conjugated secondary antibodies from Abcam mentioned previously) served as the positive control for determination of colocalization (mean Pearson's correlation coefficient = 0.93).

Techniques: Control, Quantitative Proteomics, Stable Transfection, Transfection, Plasmid Preparation, Sequencing, Labeling, Multiplex sample analysis, Western Blot, Software, Mass Spectrometry, Cell Culture

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Characterization of the AGR2 Interactome Uncovers New Players of Protein Disulfide Isomerase Network in Cancer Cells

doi: 10.1016/j.mcpro.2021.100188

Figure Lengend Snippet: Annotation of AGR2 protein–protein complexes in relation to cell signaling. Top 20 protein–protein interacting partners of AGR2 (log2FC >0; q < 0.05) identified in ( A ) T47D cells using LFQ and ( B ) H1299–AGR2 cells using SILAC quantitation. See and for source data for A and B , respectively. Proteins in red are members of the PDI family, proteins in blue are not members. C, 151 and 22 proteins (log2FC >0; q < 0.05) were identified as AGR2-interacting partners in T47D and H1299–AGR2 cells, respectively. Comparison of these proteins between cell lines has selected 16 overlapping proteins ( supplemental Table S1 ). PDI members are highlighted in red . D, Cytoscape ClueGO analysis (based on GO and KEGG pathway databases) of 16 overlapping proteins ( supplemental Table S1 ) has revealed a clear connection of proteins to the ER and ER processes. Enriched GO and KEGG terms are represented by nodes, and protein overlap between the terms is displayed by edges. Similarly, analysis of 16 overlapping proteins ( supplemental Table S1 ) with ConsensusPathDB-human tool with GO and KEGG databases clearly showed strong connection to ( E ) biological processes running in ER highlighted in blue box and to ( F ) molecular functions associated with protein translation and folding in ER, all pathways with p value <0.001 as determined by ConsensusPathDB-human tool. AGR2, anterior gradient 2; ER, endoplasmic reticulum; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; LFQ, label-free quantitation; log2FC, log2 fold change; PDI, protein disulfide isomerase; SILAC, stable isotope labeling with amino acids in cell culture.

Article Snippet: In contrast, cells labeled with two different fluorochromes for AGR2 (obtained by anti-AGR2 mouse Ab from Abnova and anti-AGR2 rabbit Ab from Abcam followed by fluorochrome-conjugated secondary antibodies from Abcam mentioned previously) served as the positive control for determination of colocalization (mean Pearson's correlation coefficient = 0.93).

Techniques: Multiplex sample analysis, Quantitation Assay, Comparison, Quantitative Proteomics, Cell Culture

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Characterization of the AGR2 Interactome Uncovers New Players of Protein Disulfide Isomerase Network in Cancer Cells

doi: 10.1016/j.mcpro.2021.100188

Figure Lengend Snippet: AGR2, PDIA3, and PDIA6 protein levels in E7 (AGR2-specific peptide) native eluates from DSP-crosslinked H1299 cells stably transfected with AGR2 gene compared with F4 (control) native eluates from the same cells

Article Snippet: In contrast, cells labeled with two different fluorochromes for AGR2 (obtained by anti-AGR2 mouse Ab from Abnova and anti-AGR2 rabbit Ab from Abcam followed by fluorochrome-conjugated secondary antibodies from Abcam mentioned previously) served as the positive control for determination of colocalization (mean Pearson's correlation coefficient = 0.93).

Techniques: Stable Transfection, Transfection, Control, Sequencing, Molecular Weight

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Characterization of the AGR2 Interactome Uncovers New Players of Protein Disulfide Isomerase Network in Cancer Cells

doi: 10.1016/j.mcpro.2021.100188

Figure Lengend Snippet: Immunofluorescence microscopy of PDIA3 and PDIA6 in relation to AGR2. Immunofluorescence staining of AGR2 ( red ) in parallel with ( A ) PDIA3 ( green ) and ( B ) PDIA6 ( green ). Merged images show colocalization of these proteins indicating the presence of AGR2–PDIA3 and AGR2–PDIA6 complexes. Nucleic staining ( blue ) was done by Hoechst 33342. The scale bar represents 10 μm. Colocalization of fluorescence signals was determined by Pearson's correlation coefficient (graphs on the right side ). See supplemental Fig. S1 for a positive colocalization control. AGR2, anterior gradient 2; PDIA3, protein disulfide isomerase A3.

Article Snippet: In contrast, cells labeled with two different fluorochromes for AGR2 (obtained by anti-AGR2 mouse Ab from Abnova and anti-AGR2 rabbit Ab from Abcam followed by fluorochrome-conjugated secondary antibodies from Abcam mentioned previously) served as the positive control for determination of colocalization (mean Pearson's correlation coefficient = 0.93).

Techniques: Immunofluorescence, Microscopy, Staining, Fluorescence, Control

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Characterization of the AGR2 Interactome Uncovers New Players of Protein Disulfide Isomerase Network in Cancer Cells

doi: 10.1016/j.mcpro.2021.100188

Figure Lengend Snippet: Validation of PDIA3 and PDIA6 as AGR2-interacting partners. The combined procedures of IP and SDS-PAGE were used in complex protein mixtures from T47D cells either exposed or unexposed to DSP in order to ( A ) precipitate AGR2 by specific antibody (s); ( B ) precipitate PDIA3 ( left part ) and PDIA6 ( right part ) by specific antibodies (s). Nonspecific antibody (ns) served as a negative control ( third line ). C, PLA images of complexes AGR2–PDIA3/6: red signals emerge only when proteins are closely localized. Nucleic staining ( blue ) was done by DAPI. The scale bar represents 20 μm. See supplemental Fig. S5 for corresponding PLA results in H1299 and A549 cell lines. AGR2, anterior gradient 2; DAPI, 4′,6-diamidino-2-phenylindole; IP, immunoprecipitation; PDIA, protein disulfide isomerase A; PLA, proximity ligation assay.

Article Snippet: In contrast, cells labeled with two different fluorochromes for AGR2 (obtained by anti-AGR2 mouse Ab from Abnova and anti-AGR2 rabbit Ab from Abcam followed by fluorochrome-conjugated secondary antibodies from Abcam mentioned previously) served as the positive control for determination of colocalization (mean Pearson's correlation coefficient = 0.93).

Techniques: Biomarker Discovery, SDS Page, Negative Control, Staining, Immunoprecipitation, Proximity Ligation Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Characterization of the AGR2 Interactome Uncovers New Players of Protein Disulfide Isomerase Network in Cancer Cells

doi: 10.1016/j.mcpro.2021.100188

Figure Lengend Snippet: The effect of ER inducers on AGR2–PDIA3 complex formation. Changes in subcellular localization of AGR2–PDIA3 complex in response to tunicamycin (TUN) and thapsigargin (THG) in comparison with untreated (control, CTR) ( A ) T47D and ( B ) A549 cells were analyzed using immunofluorescence staining for AGR2 ( green ), PDIA3 ( red ), and nucleus by DAPI ( blue ). The scale bar represents 10 μm. Colocalization of fluorescence signals was determined by Pearson's correlation coefficient, nonparametric one-way ANOVA (Kruskal–Wallis test with Dunn correction) test was used to calculate the statistical significance, ∗∗∗ p ≤ 0.001. AGR2, anterior gradient 2; DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; ns, nonsignificant; PDIA3, protein disulfide isomerase A3.

Article Snippet: In contrast, cells labeled with two different fluorochromes for AGR2 (obtained by anti-AGR2 mouse Ab from Abnova and anti-AGR2 rabbit Ab from Abcam followed by fluorochrome-conjugated secondary antibodies from Abcam mentioned previously) served as the positive control for determination of colocalization (mean Pearson's correlation coefficient = 0.93).

Techniques: Comparison, Control, Immunofluorescence, Staining, Fluorescence

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Characterization of the AGR2 Interactome Uncovers New Players of Protein Disulfide Isomerase Network in Cancer Cells

doi: 10.1016/j.mcpro.2021.100188

Figure Lengend Snippet: ER stress induces complex formation followed by enhanced secretion of AGR2. Immunochemical analysis of ( A ) intracellular and ( B ) extracellular AGR2 and PDIA3 in response to induction of ER stress. The numbers under the boxes represent relative fold changes in absorbance reflecting protein levels normalized on intracellular actin density of treated cells in relation to serum-starved cells (serum-free media [SFM]). C, IP of AGR2–PDIA3 complexes followed by SDS-PAGE in cells exposed to different inducers of ER stress. D, subcellular protein fractionation of several ER-resident proteins in A549 and T47D cells treated with ER stress inducers. The numbers under the boxes represent fold changes in absorbance reflecting protein levels normalized on GAPDH density of treated cells in relation to DMSO-exposed cells. PDIA3∗ represents the same experiment however, with prolonged exposition time to show redistribution of PDIA3 to the cytosol. Each experiment was performed at least three times. Average fold changes along with standard deviations are shown in supplemental Fig. S6 . AGR2, anterior gradient 2; CF, cytosolic fraction; DMSO, dimethyl sulfoxide; ER, membrane bound fraction containing endoplasmic reticulum; IP, immunoprecipitation; PDIA3, protein disulfide isomerase A3; WL, whole lysate.

Article Snippet: In contrast, cells labeled with two different fluorochromes for AGR2 (obtained by anti-AGR2 mouse Ab from Abnova and anti-AGR2 rabbit Ab from Abcam followed by fluorochrome-conjugated secondary antibodies from Abcam mentioned previously) served as the positive control for determination of colocalization (mean Pearson's correlation coefficient = 0.93).

Techniques: SDS Page, Fractionation, Membrane, Immunoprecipitation

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Characterization of the AGR2 Interactome Uncovers New Players of Protein Disulfide Isomerase Network in Cancer Cells

doi: 10.1016/j.mcpro.2021.100188

Figure Lengend Snippet: Binding interface classification of the top 10 solutions from monomer and dimer AGR2 docking experiments to PDIA3

Article Snippet: In contrast, cells labeled with two different fluorochromes for AGR2 (obtained by anti-AGR2 mouse Ab from Abnova and anti-AGR2 rabbit Ab from Abcam followed by fluorochrome-conjugated secondary antibodies from Abcam mentioned previously) served as the positive control for determination of colocalization (mean Pearson's correlation coefficient = 0.93).

Techniques: Binding Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Characterization of the AGR2 Interactome Uncovers New Players of Protein Disulfide Isomerase Network in Cancer Cells

doi: 10.1016/j.mcpro.2021.100188

Figure Lengend Snippet: Visualization of the interaction between AGR2 monomer and PDIA3. A representative of the best docking solution from GalaxyHeteromer experiment for monomeric AGR2 ( yellow ) is visualized in complex with PDIA3 ( gray ). N termini (Ile36) and C termini (Leu175) of ARG2 are indicated in light and purple solid van der Waals radii spheres, respectively. N termini (Ser25) and C termini (Glu493) of PDIA3 are indicated in light and dark blue solid van der Waals radii spheres , respectively. PDIA3 domains are labeled according to the description of the PDB file and following this legend: a-domain comprises residues Ser25–Gly133; b-domain comprises residues Pro134–Gly242; b'-domain comprises residues Ile243–Lys366; and a'-domain comprises residues Ser367–Ala484. Active site motifs are shown in red for both proteins; their sequences and residue numbers are indicated in proximity and with the same color code. AGR2, anterior gradient 2; PDB, Protein Data Bank; PDIA3, protein disulfide isomerase A3.

Article Snippet: In contrast, cells labeled with two different fluorochromes for AGR2 (obtained by anti-AGR2 mouse Ab from Abnova and anti-AGR2 rabbit Ab from Abcam followed by fluorochrome-conjugated secondary antibodies from Abcam mentioned previously) served as the positive control for determination of colocalization (mean Pearson's correlation coefficient = 0.93).

Techniques: Labeling, Residue

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Characterization of the AGR2 Interactome Uncovers New Players of Protein Disulfide Isomerase Network in Cancer Cells

doi: 10.1016/j.mcpro.2021.100188

Figure Lengend Snippet: AGR2-interacting proteins overlapped in three independent studies (( 54 , 55 ) and ours)

Article Snippet: In contrast, cells labeled with two different fluorochromes for AGR2 (obtained by anti-AGR2 mouse Ab from Abnova and anti-AGR2 rabbit Ab from Abcam followed by fluorochrome-conjugated secondary antibodies from Abcam mentioned previously) served as the positive control for determination of colocalization (mean Pearson's correlation coefficient = 0.93).

Techniques: