Journal: Gastroenterology

Article Title: Loss of Trefoil Factor 2 From Pancreatic Duct Glands Promotes Formation of Intraductal Papillary Mucinous Neoplasms in Mice

doi: 10.1053/j.gastro.2016.07.045

Figure Lengend Snippet: PDG comprise the basal segment of SB-IPMN in humans. (A) The cyst walls of SB-IPMN demonstrate expansion and crowding of hyperplastic PDG (left) PDG are identified by MUC6 (green) and TFF2 (yellow). They are seen to fuse (asterisk) and open into the IPMN cyst wall (arrow). PDG are also found in the bottom layer/crypts between each papillary structure of IPMN (right). Scale Bars: 100 µm. (B) Quantification of TFF2 expression in IPMN (n=13) and PDAC (n=15). (C) Three different histologic patterns of PDG (arrow) within SB-IPMN. The hyperplastic phase (left), the cystic metaplasia phase (middle) and the papillary phase (right). Each PDG can be identified by its expression of TFF2, and Ki-67-positive proliferating cells are found in the PDG compartment (bottom). Scale Bars: 100 µm.

Article Snippet: Recombinant Human TFF2 protein from R&D Systems (catalogue # 8290-TF) and transfected in PANC-1 and Aspc-1 cells as per published report 26 .

Techniques: Expressing

Journal: Gastroenterology

Article Title: Loss of Trefoil Factor 2 From Pancreatic Duct Glands Promotes Formation of Intraductal Papillary Mucinous Neoplasms in Mice

doi: 10.1053/j.gastro.2016.07.045

Figure Lengend Snippet: Human IPMN are comprised of multiple PDG/IPMN units. The dotted frame outlines three PDG/IPMN units. (A, B, C) Proliferation (Ki-67; arrow) occurs in a narrow zone located between the TFF2-positive PDG and the overlying IPMN. Scale Bars: 50 µm (D) Within each PDG/IPMN unit, Ki-67-positive PDG cells and their overlying IPMN epithelia were isolated by LCM. Scale Bars: 50 µm (E) The D-loops of mitochondrial DNA reveal the same mutational profile (arrow) in each PDG/IPMN unit.

Article Snippet: Recombinant Human TFF2 protein from R&D Systems (catalogue # 8290-TF) and transfected in PANC-1 and Aspc-1 cells as per published report 26 .

Techniques: Isolation

Journal: Gastroenterology

Article Title: Loss of Trefoil Factor 2 From Pancreatic Duct Glands Promotes Formation of Intraductal Papillary Mucinous Neoplasms in Mice

doi: 10.1053/j.gastro.2016.07.045

Figure Lengend Snippet: Loss of TFF2 accelerates tumorization of KC mice. (A) While KC mice develop pseudopapillary lesions in the main pancreatic duct by 4 months, KC/TFF2KO mice show large papillary structures with increased PDG in both size and number (arrow) by 2 months. Scale Bars: 100 µm (B) Size, number and BrdU-positive PDG increase in both KC/TFF2+/− and KC/TFF2−/− mice (p<0.05). (C) Pseudo-papillary lesions in KC mice (left), low-grade papillary structure in KC/TFF2+/− mice (middle), high-grade papillary structure in KC/TFF2−/− mice (right). Scale Bars: 50 µm (D) Papillary structure in KC/TFF2−/− mice express gastric mucins MUC5AC and MUC6. BrdU is incorporated in PDG compartment. Scale Bars: 50 µm.

Article Snippet: Recombinant Human TFF2 protein from R&D Systems (catalogue # 8290-TF) and transfected in PANC-1 and Aspc-1 cells as per published report 26 .

Techniques:

Journal: Gastroenterology

Article Title: Loss of Trefoil Factor 2 From Pancreatic Duct Glands Promotes Formation of Intraductal Papillary Mucinous Neoplasms in Mice

doi: 10.1053/j.gastro.2016.07.045

Figure Lengend Snippet: Carcinogenesis in KC/TFF2KO mice at the age of 6 months. (A) While KC mice show only mPanIN-1 (left), KC/TFF2KO mice show mPanIN-2 (middle) and mPanIN-3 (right). Scale Bars: 200 µm (top) and 50 µm (bottom) (B) The PanIN-occupied area is significantly larger in TFF2-dificient mice (p<0.01). (C) A KC/TFF2+/− mice was found to have PDAC in the pancreatic head (top, arrowheads) with multiple liver (top, white arrows: bottom, left, black arrows) and lung metastases (bottom, right, black arrows). Scale Bars: 50 µm.

Article Snippet: Recombinant Human TFF2 protein from R&D Systems (catalogue # 8290-TF) and transfected in PANC-1 and Aspc-1 cells as per published report 26 .

Techniques:

Journal: Gastroenterology

Article Title: Loss of Trefoil Factor 2 From Pancreatic Duct Glands Promotes Formation of Intraductal Papillary Mucinous Neoplasms in Mice

doi: 10.1053/j.gastro.2016.07.045

Figure Lengend Snippet: TFF2 inhibits cell-proliferation via SMAD4 in vitro. (A) RNA expression of TFF2 in HPDE and cancer cell lines (Real-Time PCR). (B) Growth curve showing TFF2 dose-dependent inhibitory effects on proliferation. (C) Overexpression of TFF2 induced upregulation of SMAD4. (D) SMAD4 expression can be found in nuclei after the overexpression of TFF2. (E) Double-positive cells for TFF2 and BrdU can be found after the suppression of SMAD4. (F) The downregulation of proliferation by TFF2 can be restored by the SMAD4.

Article Snippet: Recombinant Human TFF2 protein from R&D Systems (catalogue # 8290-TF) and transfected in PANC-1 and Aspc-1 cells as per published report 26 .

Techniques: In Vitro, RNA Expression, Real-time Polymerase Chain Reaction, Over Expression, Expressing

Journal: Gastroenterology

Article Title: Loss of Trefoil Factor 2 From Pancreatic Duct Glands Promotes Formation of Intraductal Papillary Mucinous Neoplasms in Mice

doi: 10.1053/j.gastro.2016.07.045

Figure Lengend Snippet: TFF2 promoter methylation and SMAD4 regulation in vitro. (A) TFF2 promoter DNA methylation profiles of PANC-1 and Aspc-1 cells. Lymphocyte DNA and Sss1 methylated DNA are used as controls. (B) TFF2 gene is upregulated following the genomic demethylation by decitabine. (C) After treatment with decitabine, promoter methylation in all the 5 CpG sites was decreased. (D) Treatment with decitabine upregulated TFF2 and SMAD4 mRNA. However, siRNA-mediated knockdown of TFF2 abrogated the decitabine-mediated SMAD4 upregulation.

Article Snippet: Recombinant Human TFF2 protein from R&D Systems (catalogue # 8290-TF) and transfected in PANC-1 and Aspc-1 cells as per published report 26 .

Techniques: Methylation, In Vitro, DNA Methylation Assay, Knockdown

Journal: Scientific Reports

Article Title: Nardilysin regulates inflammation, metaplasia, and tumors in murine stomach

doi: 10.1038/srep43052

Figure Lengend Snippet: ( A ) Representative macroscopic views of the stomachs of Nrdc + / + and Nrdc −/− mice. ( B ) H&E staining of Nrdc + / + and Nrdc −/− mouse stomachs. Bars = 100 μm. ( C ) Immunohistochemistry for pepsinogen II, H + /K + -ATPase, Muc5ac, TFF2, and Ki67 in Nrdc + / + and Nrdc −/− mice. Bars = 100 μm. ( D ) Percentages of epithelial cells immunostained with pepsinogen II, H + /K + -ATPase, Muc5ac, TFF2, and Ki67 in Nrdc + / + and Nrdc −/− mice.

Article Snippet: The primary antibodies used were rat anti-F4/80 (Abcam, Cambridge, MA, USA), rat anti–Gr-1 (eBioscience, San Diego, CA, USA), sheep anti-pepsinogen II (Abcam), mouse anti-H + /K + -ATPase α subunit (MBL, Nagoya, Japan), mouse anti-Muc5AC (Abcam), mouse-anti-spasmolytic polypeptide (TFF2) (R&D Systems, Minneapolis, MN, USA), and rat anti-Ki67 (Dako, Glostrup, Denmark).

Techniques: Staining, Immunohistochemistry

Journal: Scientific Reports

Article Title: Nardilysin regulates inflammation, metaplasia, and tumors in murine stomach

doi: 10.1038/srep43052

Figure Lengend Snippet: ( A )Immunohistochemistry for TFF2 in Nrdc + / + and Nrdc −/− mouse stomachs with Helicobacter felis infection. Bars = 100 μm. ( B ) Areas stained for TFF2 in Nrdc + / + and Nrdc −/− mouse stomachs with Helicobacter felis infection. *P < 0.05. ( C ) Alcian blue staining of Nrdc + / + and Nrdc −/− mouse stomachs with Helicobacter felis infection. Bars = 100 μm. ( D ) Areas stained with Alcian blue in Nrdc + / + and Nrdc −/− mouse stomachs with Helicobacter felis infection. *P < 0.05. ( E ) Alcian blue staining of Nrdc + / + and Nrdc −/− mouse stomachs with PGE 2 expression. Bars = 1000 μm. ( D ) Areas stained with Alcian blue in Nrdc + / + and Nrdc −/− mouse stomachs with forced PGE 2 expression. *P < 0.05.

Article Snippet: The primary antibodies used were rat anti-F4/80 (Abcam, Cambridge, MA, USA), rat anti–Gr-1 (eBioscience, San Diego, CA, USA), sheep anti-pepsinogen II (Abcam), mouse anti-H + /K + -ATPase α subunit (MBL, Nagoya, Japan), mouse anti-Muc5AC (Abcam), mouse-anti-spasmolytic polypeptide (TFF2) (R&D Systems, Minneapolis, MN, USA), and rat anti-Ki67 (Dako, Glostrup, Denmark).

Techniques: Immunohistochemistry, Infection, Staining, Expressing

Journal: The Journal of Physiology

Article Title: Trefoil factor 2 activation of CXCR4 requires calcium mobilization to drive epithelial repair in gastric organoids

doi: 10.1113/JP277259

Figure Lengend Snippet: Results from WT and TFF2 KO gastric organoids imaging over time, measuring the movement of fluorescent nuclei (Hoechst 33342 stain) after PD. PD occurred at t = 0 min. WT and TFF2 KO gastric organoids were treated with AMD3100 (1 μm) for 1 h or BAPTA/AM (50 μm) for 30 min before PD as indicated. rTFF2 was microinjected into the lumen of organoids 30 min before the study (see Methods). Exfoliation was determined based on the maximum distance of damaged nuclei into gastric organoid lumen over 20 min. Vehicle (WT control, n = 7; TFF2 KO control, n = 10; TFF2 + rTFF2 Control, n = 10); AMD3100 (WT, n = 6; TFF2 KO, n = 6; TFF2 KO + rTFF2, n = 5); BAPTA/AM (WT, n = 4; TFF2 KO, n = 4; TFF2 KO + rTFF2, n = 8). * P < 0.05 vs. WT vehicle, #P < 0.05 vs. rTFF2 treatment in TFF2 KO.

Article Snippet: For rescue experiments in TFF2 and NHE2 KO gastric organoids, recombinant human TFF2 (rTFF2; 40 μ m stock; R&D Systems, Minneapolis, MN, USA) was microinjected as described previously (Engevik et al . 2018 ).

Techniques: Imaging, Staining, Control

Journal: The Journal of Physiology

Article Title: Trefoil factor 2 activation of CXCR4 requires calcium mobilization to drive epithelial repair in gastric organoids

doi: 10.1113/JP277259

Figure Lengend Snippet: Results from imaging of WT and TFF2 KO organoids over time; measuring the movement of fluorescent nuclei (Hoechst 33342 stain) after PD. Some organoids were treated with AG1478 (200 nm) as indicated. rTFF2 was microinjected into the lumen of organoids before the study. Exfoliation was determined based on the maximum distance of damaged nuclei into gastric organoid lumen over 20 min. Vehicle (WT control, n = 6; TFF2 KO control, n = 8; TFF2 KO + rTFF2, n = 8); AG1478 (WT, n = 6; TFF2 KO, n = 5; TFF2 KO + rTFF2, n = 5). * P < 0.05 vs. WT vehicle, #P < 0.05 vs. rTFF2 treatment in TFF2 KO.

Article Snippet: For rescue experiments in TFF2 and NHE2 KO gastric organoids, recombinant human TFF2 (rTFF2; 40 μ m stock; R&D Systems, Minneapolis, MN, USA) was microinjected as described previously (Engevik et al . 2018 ).

Techniques: Imaging, Staining, Control

Journal: The Journal of Physiology

Article Title: Trefoil factor 2 activation of CXCR4 requires calcium mobilization to drive epithelial repair in gastric organoids

doi: 10.1113/JP277259

Figure Lengend Snippet: Fluorescence of YC‐Nano gastric organoids imaged over time in (A) to (D) and cell exfoliation measured over time in (E) to (F). Where indicated, Hoe 694 (100 μm) was added to organoid medium 1 h prior to experimentation. In time courses, PD occurred at t = 0 min. A, damage area measured in YC‐Nano control (black) and Hoe 694 supplemented gastric organoids (white) (n = 4). B, comparison of rate of repair between YC‐Nano control (black) and Hoe 694 supplemented gastric organoids (white) (* P < 0.05). C, measurement of the normalized FRET/CFP ratio of the lateral membrane region of cells adjacent to the damage site comparing control (black) and Hoe 694 supplemented gastric organoids (white). D, comparison of the maximum FRET/CFP ratio from (C) between control (black) and Hoe 694 supplemented gastric organoids (white) (n = 4, * P < 0.05). E, comparison of exfoliation in WT (n = 5) and NHE2 KO vehicle (n = 5) and rTFF2 injected organoids (n = 6) (* P < 0.05). F, comparison of exfoliation in WT and TFF2 KO gastric organoids treated with Hoe 694 and/or microinjection of rTFF2. Vehicle (WT Control, n = 5; TFF2 KO, n = 6; TFF2 KO + rTFF2, n = 4); Hoe 694 (WT, n = 5; TFF2 KO, n = 4; TFF2 KO + rTFF2, n = 4). * P < 0.05 vs. WT vehicle, #P < 0.05 vs. rTFF2 treatment in TFF2 KO.

Article Snippet: For rescue experiments in TFF2 and NHE2 KO gastric organoids, recombinant human TFF2 (rTFF2; 40 μ m stock; R&D Systems, Minneapolis, MN, USA) was microinjected as described previously (Engevik et al . 2018 ).

Techniques: Fluorescence, Control, Comparison, Membrane, Injection, Microinjection

Journal: Digestive diseases and sciences

Article Title: Pathophysiological investigation of the gastric surface mucous gel layer of patients with Helicobacter pylori infection by using immunoassays for trefoil factor family 2 and gastric gland mucous cell-type mucin in gastric juice.

doi: 10.1007/s10620-011-1724-9

Figure Lengend Snippet: Fig. 2 Heterogeneity of the trefoil factor family 2 molecule in gastric juice, and its influence on the present assay method. a Recombinant trefoil factor family 2 (TFF2) (lane Ag) and patients’ gastric juices (lanes 1–4), treated with Laemmli buffer (without 2-mercap- toethanol), were loaded onto 4–20% gradient polyacrylamide gels. After electrophoresis, separated proteins were transferred electropho- retically onto a nitrocellulose membrane. The bands containing TFF2 were visualized by immunoblot analysis using anti-TFF2 antibody. b To investigate the effect of heterogeneity of the TFF2 molecule, we measured TFF2 levels in the serial dilutions of mixed-gastric juice samples that were prepared using two gastric juices (lanes 2 and 4 in a). Data are expressed as mean ± standard deviation (SD) derived from triplicate determinations in each of the two separate experiments

Article Snippet: The biotinylated anti-human TFF2 antibody was prepared by using the goat polyclonal anti-human TFF2 antibody (R&D systems) and the EZ-Link Sulfo-NHS-LC-Biotinylation kit (Pierce, Rockford, IL) according to the manufacturer’s instructions.

Techniques: Recombinant, Electrophoresis, Membrane, Western Blot, Standard Deviation, Derivative Assay

Journal: Digestive diseases and sciences

Article Title: Pathophysiological investigation of the gastric surface mucous gel layer of patients with Helicobacter pylori infection by using immunoassays for trefoil factor family 2 and gastric gland mucous cell-type mucin in gastric juice.

doi: 10.1007/s10620-011-1724-9

Figure Lengend Snippet: Fig. 3 TFF2 concentration in gastric juices from healthy individuals and patients. a TFF2 concentration in gastric juices from patients with gastritis, gastric ulcer (GU), duodenal ulcer (DU) and healthy individuals (control). b TFF2 concentration in gastric juices from patients with H. pylori infection (HP (?)) and in H. pylori-negative patients (HP (-)). c TFF2 concentration in gastric juices from patients before and after the H. pylori eradication. *p \ 0.05; **p \ 0.01; NS not significant

Article Snippet: The biotinylated anti-human TFF2 antibody was prepared by using the goat polyclonal anti-human TFF2 antibody (R&D systems) and the EZ-Link Sulfo-NHS-LC-Biotinylation kit (Pierce, Rockford, IL) according to the manufacturer’s instructions.

Techniques: Concentration Assay, Control, Infection

Journal: PLoS ONE

Article Title: Increased trefoil factor 2 levels in patients with chronic kidney disease

doi: 10.1371/journal.pone.0174551

Figure Lengend Snippet: Panel A: TFF2 serum levels, one data point outside the axis limits in early CKD stages. Panel B: TFF2 urine concentrations. Panel C: Fractional TFF2 excretion, one data point outside the axis limits in later CKD stages. Each dot represents an individual patient. The line indicates the median. CKD, chronic kidney disease. Only significant p-values are given.

Article Snippet: TFF2 concentrations were measured by an enzyme-linked immunosorbent assay (ELISA) kit (Human TFF2 DuoSet, R&D Systems, Minneapolis, Minnesota), based on Vestergaard et al [ ].

Techniques:

Journal: PLoS ONE

Article Title: Increased trefoil factor 2 levels in patients with chronic kidney disease

doi: 10.1371/journal.pone.0174551

Figure Lengend Snippet: TFF2 serum and urine concentrations as well as fract TFF2 excretion analysed within nephropathies diagnosed in more than 10 patients.

Article Snippet: TFF2 concentrations were measured by an enzyme-linked immunosorbent assay (ELISA) kit (Human TFF2 DuoSet, R&D Systems, Minneapolis, Minnesota), based on Vestergaard et al [ ].

Techniques:

Journal: PLoS ONE

Article Title: Increased trefoil factor 2 levels in patients with chronic kidney disease

doi: 10.1371/journal.pone.0174551

Figure Lengend Snippet: Correlation of TFF2 serum and urine concentrations with clinical and kidney function parameters.

Article Snippet: TFF2 concentrations were measured by an enzyme-linked immunosorbent assay (ELISA) kit (Human TFF2 DuoSet, R&D Systems, Minneapolis, Minnesota), based on Vestergaard et al [ ].

Techniques:

Journal: PLoS ONE

Article Title: Increased trefoil factor 2 levels in patients with chronic kidney disease

doi: 10.1371/journal.pone.0174551

Figure Lengend Snippet: Panel A: Serum TFF2 and serum creatinine correlated significantly using Spearman's rank correlation coefficient (Spearman's r = 0.44, p < 0.001, 113 pairs). The X and the Y-axis are given as log scale. Panel B: Urine TFF2 and serum creatinine negatively correlated using Spearman's rank correlation coefficient (Spearman's r = 0.4, p < 0.001, 111 pairs). The X and the Y-axis are given as log scale.

Article Snippet: TFF2 concentrations were measured by an enzyme-linked immunosorbent assay (ELISA) kit (Human TFF2 DuoSet, R&D Systems, Minneapolis, Minnesota), based on Vestergaard et al [ ].

Techniques:

Journal: PLoS ONE

Article Title: Increased trefoil factor 2 levels in patients with chronic kidney disease

doi: 10.1371/journal.pone.0174551

Figure Lengend Snippet: Panel A: ROC curve for serum TFF2 and later CKD stages, AUC 0.79 (0.70–0.88, p < 0.001). Panel B: ROC curve for urine TFF2 and early CKD stages, AUC 0.75 (0.63–0.87, p < 0.001).

Article Snippet: TFF2 concentrations were measured by an enzyme-linked immunosorbent assay (ELISA) kit (Human TFF2 DuoSet, R&D Systems, Minneapolis, Minnesota), based on Vestergaard et al [ ].

Techniques: