Journal: International journal of molecular sciences

Article Title: Slowed Intestinal Transit Induced by Less Mucus in Intestinal Goblet Cell Piezo1-Deficient Mice through Impaired Epithelial Homeostasis.

doi: 10.3390/ijms241814377

Figure Lengend Snippet: Figure 1. Identification of Piezo1∆GC mice. (A) Immunofluorescence co-localization of Piezo1 and Agr2 (label goblet cells) in WT and Piezo1∆GC mouse colons. The yellow box shows a magnified localized image. Scale bar: 100 µm. (B) mRNA level of Piezo1 in WT and Piezo1∆GC mouse colon crypts (normalized to GAPDH). At least three independent experiments were conducted. Data are expressed as the mean ± SEM. (n = 5 mice). ** p < 0.01.

Article Snippet: The antibodies used in our experiments included the following: Piezo1 (1:200, 15939-1-AP, Proteintech, Chicago, IL, USA), Agr2 (1:200, AF6068, R & D Systems, Minnesota, USA), Ki67 (1:200, GB111141, Servicebio, Wuhan, China), cKit (1:150, ab256345, Abcam, Cambridge, England), Alpi (1:200, A0514, Abclonal, Woburn, MA, USA), and Chga (1:200, A9576, Abclonal).

Techniques:

Journal: International journal of molecular sciences

Article Title: Slowed Intestinal Transit Induced by Less Mucus in Intestinal Goblet Cell Piezo1-Deficient Mice through Impaired Epithelial Homeostasis.

doi: 10.3390/ijms241814377

Figure Lengend Snippet: Figure 2. Decreased GC numbers and thinner mucus layer in Piezo1∆GC mouse colons. (A) Im- munofluorescence staining of Agr2 in WT and Piezo1∆GC mouse colons. Scale bar: 100 µm. The crypt was divided equally into three parts: upper, middle, and base. The white arrow indicates a goblet cell. (B) Statistical analysis of goblet cells/epithelial cells in (A). (C) Statistical analysis of goblet cells in different parts/epithelial cells in (A). (D) RNA level of Mucin2 in colon tissues from WT and Piezo1∆GC mice (normalized to GAPDH). (E) AB-PAS staining of mucus in WT and Piezo1∆GC mouse colons. The red arrows indicate the mucus layer. Scale bar: 50 µm. (F) Statistical analysis of mucus layer thickness in (D). At least three independent experiments were conducted. Data are expressed as the mean ± SEM. (n = 5 mice). ns, not significant; ** p < 0.01; *** p < 0.001.

Article Snippet: The antibodies used in our experiments included the following: Piezo1 (1:200, 15939-1-AP, Proteintech, Chicago, IL, USA), Agr2 (1:200, AF6068, R & D Systems, Minnesota, USA), Ki67 (1:200, GB111141, Servicebio, Wuhan, China), cKit (1:150, ab256345, Abcam, Cambridge, England), Alpi (1:200, A0514, Abclonal, Woburn, MA, USA), and Chga (1:200, A9576, Abclonal).

Techniques: Staining

Journal: International journal of molecular sciences

Article Title: Slowed Intestinal Transit Induced by Less Mucus in Intestinal Goblet Cell Piezo1-Deficient Mice through Impaired Epithelial Homeostasis.

doi: 10.3390/ijms241814377

Figure Lengend Snippet: Figure 6. Abnormal intestinal epithelial cell composition and impaired colon stem cell niche in Piezo1∆GC mice. (A) RNA levels of Ki67 and stem cell markers (Lgr5, Sox9, and EphB2) in WT and Piezo1∆GC mouse colon crypts (normalized to GAPDH). (B) Immunohistochemistry of Ki67 in WT and Piezo1∆GC mouse colons. Scale bar: 100 µm. (C) Statistical analysis of Ki67 positive cell ratio in (B). (D) RNA levels of stem cell niche marker (cKit), differentiated colonocyte marker (Alpi), goblet cell marker (Agr2), and enteroendocrine cell marker (Chga) in WT and Piezo1∆GC mouse colon crypts (normalized to GAPDH). (E) Immunohistochemistry of cKit in WT and Piezo1∆GC mouse colons. The white dashed lines mark the crypt borders, and the red arrow indicates a cKit-positive cell. Scale bar: 50 µm. (F) Statistical analysis of cKit-positive cell ratio in (E). (G,H) Immunofluorescence staining of Alpi (G) and Chga (H) in WT and Piezo1∆GC mouse colons. The white dashed lines mark crypt borders, and the white arrow indicates a differentiated colonocyte in (G) and an enteroendocrine cell in (H). Scale bar: 25 µm. (I,J) Statistical analysis of differentiated colonocyte ratio in (G) and enteroendocrine cell ratio in (H). At least three independent experiments were conducted. Data are presented as the mean ± SEM. (n = 5 mice). ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The antibodies used in our experiments included the following: Piezo1 (1:200, 15939-1-AP, Proteintech, Chicago, IL, USA), Agr2 (1:200, AF6068, R & D Systems, Minnesota, USA), Ki67 (1:200, GB111141, Servicebio, Wuhan, China), cKit (1:150, ab256345, Abcam, Cambridge, England), Alpi (1:200, A0514, Abclonal, Woburn, MA, USA), and Chga (1:200, A9576, Abclonal).

Techniques: Immunohistochemistry, Marker, Staining

Journal: International journal of molecular sciences

Article Title: Slowed Intestinal Transit Induced by Less Mucus in Intestinal Goblet Cell Piezo1-Deficient Mice through Impaired Epithelial Homeostasis.

doi: 10.3390/ijms241814377

Figure Lengend Snippet: Figure 7. Decreased self-renewal capacity of colon stem cells from Piezo1∆GC mice. (A) Representative images of colonoids from WT and Piezo1∆GC mice at day 5. Images of one colonoid on day 1, 3, and 5 are below, reflecting the growth process of a colonoid. Scale bar: 100 µm. (B–D) Indicators related to colonoids growth: number of buds per colonoid (B), surface area per colonoid (C), and percentage of colonoids with buds per well (D). (E) Immunofluorescence staining of EDU and Ki67 in colonoids from WT and Piezo1∆GC mice. Scale bar: 100 µm. (F) Statistical analysis of EDU and Ki67 positive cell ratio in (E). (G) Immunofluorescence staining of differentiated colonocyte (Alpi), goblet cell (Agr2), and enteroendocrine cell (Chga) in colonoids from WT and Piezo1∆GC mice. Scale bar: 100 µm. (H) Statistical analysis of Alpi, Agr2, and Chga positive cell ratio in (G). (I) RNA levels of Ki67 from WT and Piezo1∆GC mouse colonoids (normalized to GAPDH). (J) AB-PAS staining of colonoids from WT and Piezo1∆GC mice. The red arrows indicate mucus secreted by goblet cells in colonoids. Scale bar: 100 µm. At least three independent experiments were conducted. Data are presented as the mean ± SEM. (n = 5 mice). ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The antibodies used in our experiments included the following: Piezo1 (1:200, 15939-1-AP, Proteintech, Chicago, IL, USA), Agr2 (1:200, AF6068, R & D Systems, Minnesota, USA), Ki67 (1:200, GB111141, Servicebio, Wuhan, China), cKit (1:150, ab256345, Abcam, Cambridge, England), Alpi (1:200, A0514, Abclonal, Woburn, MA, USA), and Chga (1:200, A9576, Abclonal).

Techniques: 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: Journal of Translational Medicine

Article Title: SATB1 is overexpressed in metastatic prostate cancer and promotes prostate cancer cell growth and invasion

doi: 10.1186/1479-5876-11-111

Figure Lengend Snippet: SATB1 immunohistochemistry staining. A , benign prostate hyperplasia. B , prostatic carcinoma with metastasis. C , prostatic carcinoma without metastasis. D , negative control. Magnification: 400 × .

Article Snippet: The sections were blocked with 2% goat serum in 0.01 M PBS at room temperature for 1 h, then incubated with rabbit polyclonal anti-SATB1 antibody (1:500 dilution, NB110-17780, Novus Biologicals, Littleton, CO, USA) overnight at 4°C.

Techniques: Immunohistochemistry, Staining, Negative Control

Journal: Journal of Translational Medicine

Article Title: SATB1 is overexpressed in metastatic prostate cancer and promotes prostate cancer cell growth and invasion

doi: 10.1186/1479-5876-11-111

Figure Lengend Snippet: SATB1 expression and clinicopathologic parameters of prostate cancer

Article Snippet: The sections were blocked with 2% goat serum in 0.01 M PBS at room temperature for 1 h, then incubated with rabbit polyclonal anti-SATB1 antibody (1:500 dilution, NB110-17780, Novus Biologicals, Littleton, CO, USA) overnight at 4°C.

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: SATB1 is overexpressed in metastatic prostate cancer and promotes prostate cancer cell growth and invasion

doi: 10.1186/1479-5876-11-111

Figure Lengend Snippet: SATB1 expression is correlated with the invasion ability of prostate cancer cells. A - C : The invasion of LNCaP, DU-145 and PC-3 cells was measured by Transwell chamber. A , DU-145. B , PC-3. C , LNCaP. Magnification: 200×. D : Western blot analysis of the relative protein level of SATB1 in LNCaP, DU-145 and PC-3 cells (n = 3). β-actin served as loading control. *P < 0.05 compared to DU-145 cells. Note that SATB1 antibody could not detect SATB2 protein in the cell lysates.

Article Snippet: The sections were blocked with 2% goat serum in 0.01 M PBS at room temperature for 1 h, then incubated with rabbit polyclonal anti-SATB1 antibody (1:500 dilution, NB110-17780, Novus Biologicals, Littleton, CO, USA) overnight at 4°C.

Techniques: Expressing, Western Blot, Control

Journal: Journal of Translational Medicine

Article Title: SATB1 is overexpressed in metastatic prostate cancer and promotes prostate cancer cell growth and invasion

doi: 10.1186/1479-5876-11-111

Figure Lengend Snippet: Silencing of SATB1 in DU-145 cells. DU-145 cells were untransfected (control), transfected with pSliencer3.1 control vector or transfected with pSilencer3.1-SATB1 vector for 24 h, 48 h, or 72 h. The protein levels of SATB1 and SATB2 were detected by Western blot analysis (n = 3). β-actin served as loading control. * P < 0.05 compared to cells transfected with pSliencer3.1.

Article Snippet: The sections were blocked with 2% goat serum in 0.01 M PBS at room temperature for 1 h, then incubated with rabbit polyclonal anti-SATB1 antibody (1:500 dilution, NB110-17780, Novus Biologicals, Littleton, CO, USA) overnight at 4°C.

Techniques: Control, Transfection, Plasmid Preparation, Western Blot

Journal: Journal of Translational Medicine

Article Title: SATB1 is overexpressed in metastatic prostate cancer and promotes prostate cancer cell growth and invasion

doi: 10.1186/1479-5876-11-111

Figure Lengend Snippet: Silencing of SATB1 inhibits the invasion and growth of DU-145 cells. A - D : The invasion of DU-145 cells was measured by Transwell chamber. A, DU-145 cells untransfected. B , DU-145 cells transfected with pSliencer3.1. C , DU-145 cells transfected with pSilencer3.1-SATB1. Magnification: 200×. D : Quantitative analysis of the number of invaded cells as shown in A - C (n = 3). E : The proliferation of DU-145 cells was determined by CCK8 assay (n = 3). The cell proliferation inhibition rate was calculated for DU-145 cells at 24 h, 48 h and 72 h after transfection with pSilencer3.1-SATB1. *P < 0.05 compared to cells transfected with pSliencer3.1.

Article Snippet: The sections were blocked with 2% goat serum in 0.01 M PBS at room temperature for 1 h, then incubated with rabbit polyclonal anti-SATB1 antibody (1:500 dilution, NB110-17780, Novus Biologicals, Littleton, CO, USA) overnight at 4°C.

Techniques: Transfection, CCK-8 Assay, Inhibition

Journal: Journal of Translational Medicine

Article Title: SATB1 is overexpressed in metastatic prostate cancer and promotes prostate cancer cell growth and invasion

doi: 10.1186/1479-5876-11-111

Figure Lengend Snippet: Reconstitution of SATB1 expression in LNCaP cells. LNCaP cells were untransfected (control), transfected with pcDNA3.1 control vector or transfected with pcDNA3.1-SATB1 vector for 12 h, 24 h, or 36 h. A . The mRNA level of SATB1 was detected by RT-PCR (n = 3). B . The protein level of SATB1 was detected by Western blot analysis (n = 3). β-actin served as loading control. *P < 0.05 compared to cells transfected with pcDNA3.1.

Article Snippet: The sections were blocked with 2% goat serum in 0.01 M PBS at room temperature for 1 h, then incubated with rabbit polyclonal anti-SATB1 antibody (1:500 dilution, NB110-17780, Novus Biologicals, Littleton, CO, USA) overnight at 4°C.

Techniques: Expressing, Control, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of Translational Medicine

Article Title: SATB1 is overexpressed in metastatic prostate cancer and promotes prostate cancer cell growth and invasion

doi: 10.1186/1479-5876-11-111

Figure Lengend Snippet: Overexpression of SATB1 promotes the invasion and growth of LNCaP cells. A - D : The invasion of LNCaP cells was measured by Transwell chamber. A , LNCaP cells untransfected. B , LNCaP cells transfected with pcDNA3.1. C , LNCaP cells transfected with pcDNA3.1-SATB1. Magnification: 200×. D : Quantitative analysis of the number of invaded cells as shown in A - C (n = 3). *P < 0.05 compared to cells transfected with pcDNA3.1. E : The proliferation of LNCaP cells was determined by CCK8 assay (n = 3). The cell proliferation ratio was calculated for LNCaP cells at 24 h, 48 h and 72 h after transfection of pcDNA3.1 or pcDNA3.1-SATB1. *P < 0.05 compared to corresponding cells transfected with pcDNA3.1.

Article Snippet: The sections were blocked with 2% goat serum in 0.01 M PBS at room temperature for 1 h, then incubated with rabbit polyclonal anti-SATB1 antibody (1:500 dilution, NB110-17780, Novus Biologicals, Littleton, CO, USA) overnight at 4°C.

Techniques: Over Expression, Transfection, CCK-8 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 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