Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Deficiency of Stomach-Type Claudin-18 in Mice Induces Gastric Tumor Formation Independent of H pylori Infection

doi: 10.1016/j.jcmgh.2019.03.003

Figure Lengend Snippet: Antibodies Used in Immunofluorescence Images and FACS Analysis

Article Snippet: TFF2 , Bioss Antibodies , bs-1921R , Unconjugate.

Techniques: Immunofluorescence

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: Figure 2. PDG comprise the basal segment of SB- IPMN in humans. (A) The cyst walls of SB-IPMN demonstrate expansion and crowding of hyper- plastic 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 ¼ 100 mm. (B) Quantification of TFF2 expression in IPMN (n ¼ 13) and PDAC (n ¼ 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 identi- fied by its expression of TFF2, and Ki-67–positive proliferating cells are found in the PDG compartment (bottom). Scale bars ¼ 100 mm.

Article Snippet: The protein (25mg)was subjected to 10%sodiumdodecyl sulfate polyacrylamide electrophoresis and transferred to a nitrocellulose membrane, which was incubated with primary antibodies against TFF2 (rabbit polyclonal, 1:500; Protein Tech, Chicago, IL) and SMAD4 (rabbit monoclonal, clone EP618Y, 1:1000; Abcam, Cambridge, MA) then detected using a chemiluminescence kit (Supersignal West Pico; Thermo Scientific), or visualized using an Odyssey IR Scanner (LI-COR Biosciences, Lincoln, NE) system and LI-COR secondary fluorescent antibodies.

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: Figure 3. Human IPMN are composed of multiple PDG/IPMN units. The dotted frame outlines 3 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 ¼ 50 mm (D) Within each PDG/IPMN unit, Ki- 67–positive PDG cells and their overlying IPMN epithelia were isolated by laser capture microscopy. Scale bars ¼ 50 mm. (E) The D-loops of mitochon- drial DNA reveal the same mutational profile (arrow) in each PDG/IPMN unit.

Article Snippet: The protein (25mg)was subjected to 10%sodiumdodecyl sulfate polyacrylamide electrophoresis and transferred to a nitrocellulose membrane, which was incubated with primary antibodies against TFF2 (rabbit polyclonal, 1:500; Protein Tech, Chicago, IL) and SMAD4 (rabbit monoclonal, clone EP618Y, 1:1000; Abcam, Cambridge, MA) then detected using a chemiluminescence kit (Supersignal West Pico; Thermo Scientific), or visualized using an Odyssey IR Scanner (LI-COR Biosciences, Lincoln, NE) system and LI-COR secondary fluorescent antibodies.

Techniques: Isolation, Microscopy

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: Figure 4. Loss of TFF2 accelerates tumorization of KC mice. (A) While KC mice develop pseudopa- pillary 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 ¼ 100 mm. (B) Size, number, and BrdU-positive PDG in- crease in both KC/TFF2þ/

Article Snippet: The protein (25mg)was subjected to 10%sodiumdodecyl sulfate polyacrylamide electrophoresis and transferred to a nitrocellulose membrane, which was incubated with primary antibodies against TFF2 (rabbit polyclonal, 1:500; Protein Tech, Chicago, IL) and SMAD4 (rabbit monoclonal, clone EP618Y, 1:1000; Abcam, Cambridge, MA) then detected using a chemiluminescence kit (Supersignal West Pico; Thermo Scientific), or visualized using an Odyssey IR Scanner (LI-COR Biosciences, Lincoln, NE) system and LI-COR secondary fluorescent antibodies.

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: Figure 5. 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 ¼ 200 mm (top) and 50 mm (bottom) (B) The PanIN-occupied area is significantly larger in TFF2- dificient mice (P < 01). (C) A KC/TFF2þ/ mice was found to have PDAC in the pancreatic head (top, ar- rowheads) with multiple liver (top, white arrows; bottom, left, black arrows) and lung metastases (bot- tom, right, black arrows). Scale bars ¼ 50 mm.

Article Snippet: The protein (25mg)was subjected to 10%sodiumdodecyl sulfate polyacrylamide electrophoresis and transferred to a nitrocellulose membrane, which was incubated with primary antibodies against TFF2 (rabbit polyclonal, 1:500; Protein Tech, Chicago, IL) and SMAD4 (rabbit monoclonal, clone EP618Y, 1:1000; Abcam, Cambridge, MA) then detected using a chemiluminescence kit (Supersignal West Pico; Thermo Scientific), or visualized using an Odyssey IR Scanner (LI-COR Biosciences, Lincoln, NE) system and LI-COR secondary fluorescent antibodies.

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: Figure 6. 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 ef- fects on proliferation. (C) Overexpression of TFF2 induced up-regulation of SMAD4. (D) SMAD4 expression can be found in nuclei after the over- expression of TFF2. (E) Double-positive cells for TFF2 and BrdU can be found after the suppres- sion of SMAD4. (F) The down-regulation of prolif- eration by TFF2 can be restored by the SMAD4.

Article Snippet: The protein (25mg)was subjected to 10%sodiumdodecyl sulfate polyacrylamide electrophoresis and transferred to a nitrocellulose membrane, which was incubated with primary antibodies against TFF2 (rabbit polyclonal, 1:500; Protein Tech, Chicago, IL) and SMAD4 (rabbit monoclonal, clone EP618Y, 1:1000; Abcam, Cambridge, MA) then detected using a chemiluminescence kit (Supersignal West Pico; Thermo Scientific), or visualized using an Odyssey IR Scanner (LI-COR Biosciences, Lincoln, NE) system and LI-COR secondary fluorescent antibodies.

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: Figure 7. 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 up-regulated after the genomic demethylation by decitabine. (C) After treatment with decitabine, promoter methylation in all the 5 CpG sites was decreased. (D) Treatment with decitabine up-regulated TFF2 and SMAD4 messenger RNA. However, small interfering RNA-mediated knockdown of TFF2 abrogated the decitabine-mediated SMAD4 up-regulation.

Article Snippet: The protein (25mg)was subjected to 10%sodiumdodecyl sulfate polyacrylamide electrophoresis and transferred to a nitrocellulose membrane, which was incubated with primary antibodies against TFF2 (rabbit polyclonal, 1:500; Protein Tech, Chicago, IL) and SMAD4 (rabbit monoclonal, clone EP618Y, 1:1000; Abcam, Cambridge, MA) then detected using a chemiluminescence kit (Supersignal West Pico; Thermo Scientific), or visualized using an Odyssey IR Scanner (LI-COR Biosciences, Lincoln, NE) system and LI-COR secondary fluorescent antibodies.

Techniques: Methylation, In Vitro, DNA Methylation Assay, Small Interfering RNA, Knockdown

Journal: PLoS ONE

Article Title: Targeted Deletion of Kcne2 Causes Gastritis Cystica Profunda and Gastric Neoplasia

doi: 10.1371/journal.pone.0011451

Figure Lengend Snippet: A. Left , Negative control for TFF2 staining in areas of GCP from a Kcne2 −/− mouse, using IgM isotype. Scale bar, 70 µm. Right , TFF2 staining in areas of GCP from a Kcne2 −/− mouse, using IgG isotype. Scale bar, 50 µm. B. TFF2 staining in cystic region of gastric mucosa from a Kcne2 −/− mouse. Scale bar, 50 µm. C. Increased magnification views of TFF2 staining in gastric mucosa from a Kcne2 −/− mouse. Left , gastric gland cross-section; right , gastric gland longitudinal section. Scale bar, 50 µm.

Article Snippet: The primary antibody concentrations used were: 0.05 µg/ml (rabbit polyclonal anti-Ki67, Vector Labs); 1 µg/ml (mouse monoclonal anti-CK-7; Abcam); 1 µg/ml (mouse monoclonal anti-TFF2; Abcam).

Techniques: Negative Control, Staining

Journal: Journal of Virology

Article Title: Co-infection of Helicobacter pylori with Epstein-Barr virus in gastric organoids enhances cell proliferation and morphogenesis

doi: 10.1128/jvi.00928-25

Figure Lengend Snippet: In vitro generation of patient-derived gastric organoids. ( A ) Workflow of human gastric organoid generation by mechanical isolation. The collection of gastric tumor and normal epithelium tissues, gland isolation, Matrigel matrix embedding, seeding, and growth factor supplement medium were included for culture of normal gastric organoid (NGO) and tumor gastric organoid (TGO), followed by micro-injection. ( B ) Representative morphology of gastric glands isolated from tumor and normal tissues. Scale bar, 1 cm (right panel) or 100 µm (left panel). ( C ) Representative morphology of primary TGO and NGO culture generation from panel B for 8 days post-seeding. Scale bar, 200 µm. Bottom panel: the growth curve of TGOs and NGOs ( n = 3) evaluated by cross-section diameter in the first passage. ns , not significant. ( D ) Expression levels of different biomarkers in gastric adjacent tissues, TGOs, and NGOs. The total RNA extracts from TGOs and NGOs in panel C at day 3 were subjected to detection by reverse transcription PCR analysis for different biomarkers of parietal cells (Amphiregulin), endocrine cells (Chromog A), neck mucous cells (TFF2), surface mucous cells (TFF1), intestinal epithelial cells (MUC2), and intestinal specific nuclear transcription factors (CDX1, CDX2), along with GAPDH as an internal control. ( E ) Immunofluorescence analysis of TGOs and NGOs in panel C. DAPI is staining for the nuclei. TRITC phalloidin is staining for the membrane structure F-actin. Scale bar, 70 µm.

Article Snippet: Rabbit antibody to TFF1 (13734-1-AP, Proteintech) or TFF2 (136811-1-AP, Proteintech) was used in this study.

Techniques: In Vitro, Derivative Assay, Isolation, Microinjection, Expressing, Reverse Transcription, Control, Immunofluorescence, Staining, Membrane

Journal: Cancer Gene Therapy

Article Title: Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer

doi: 10.1038/s41417-018-0036-z

Figure Lengend Snippet: Identification of wild-type TFF2 and fusion TFF2-CTP-Flag protein delivered by Ad- Tff2 and Ad- Tff2 -CTP-Flag in the blood. a TFF2 level assayed by ELISA in the blood from naive wild-type and CD2- Tff2 transgenic mice and mice at time point 6 months after induction of tumorigenesis with AOM/DSS treatment. Dunn’s multiple comparisons test after one-way ANOVA test, ns, non-significant, * p < 0.05, **** p < 0.001. b Amino acid sequence of fusion protein TFF2-CTP-Flag: TFF2 amino acid sequence—bold regular, CTP—regular, S —underlined, original cysteine substituted for serine, additional amino acids residues -bold italic shadow, Flag-underlined regular, Stop—stop codon. c Schematic presentation of fusion construct Tff2 -2CTP-3Flag. d Map of GV314 vector with inserted TFF2 gene. e – j Time course of TFF2 and TFF2-CTP-Flag level after single administration of adenoviruses Ad- Tff2 and Tff2 -CTP-Flag. Tff2 -null mice were injected with 5 × 10 8 pfu of Ad- Tff2 or Ad- Tff2 -CTP-Flag and sacrificed at indicated times post infection. Time-course of Tff2 mRNA ( e ) and Tff2 -CTP-Flag mRNA ( h ) expression in the liver. Total mRNA was isolated from the liver and then Tff2 mRNA was detected by qPCR, fold of change normalized on housekeeper Hprt mRNA. For each time point 3 mice were used, graphed as mean ± SD. f , i Kinetic of TFF2 protein and TFF2-CTP-Flag fusion protein in the blood assessed by ELISA and expressed as mean ± SD at each time point, three animals per group. g , j western blot for TFF2 and TFF2-CTP-Flag in mouse serum. Five microliter of mouse serum were loaded in each lane and western blot was developed with antibody produced against C-end of TFF2 molecule, arrows indicate the position of TFF2 and TFF2-CTP-Flag with calculated sizes 12 and 25–26 kDa accordingly. k Western blot of blood sample (5 µl) from Tff2 -null mouse taken on day 7 after Ad- Tff2 -CTP-Flag administration. Western blot was developed with anti-Flag antibody; arrows indicate the positions of TFF2-CTP-Flag fusion under reduced and non-reduced conditions

Article Snippet: All blots were blocked in 5% skim milk in Tris-buffered saline (TBS) plus Tween 20 (0.05%) for 1 h. Affinity-purified primary polyclonal rabbit antibodies were produced against the C-terminus of mouse TFF2 (New England Peptide) and used at concentration 1 μg ml −1 .

Techniques: Enzyme-linked Immunosorbent Assay, Transgenic Assay, Sequencing, Construct, Plasmid Preparation, Injection, Infection, Expressing, Isolation, Western Blot, Produced

Journal: Cancer Gene Therapy

Article Title: Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer

doi: 10.1038/s41417-018-0036-z

Figure Lengend Snippet: Ad- Tff2 or Ad- Tff2 -CTP-Flag reduces splenomegaly and proportion of myeloid cells in the spleen of wild-type and Tff2 -null mice. a – f Wild-type mice were treated 2% DSS for 5 consecutive days, then on day 12 post treatment with DSS single injection of Ad- Tff2 ( a – c ) or Ad- Tff2 -CTP ( d – f ) and Ad-Fc or Ad-Fc-CTP-Flag as controls. Dose 5 × 10 8 pfu was administered in tail vein. Mice were sacrificed on day 19. Spleen appearance ( a , d ), spleen size mass ( b , e ) and proportion of CD11b + Gr-1 + cells ( c, f ) in spleen, unpaired t -test, * p < 0.05. Two experiments with three mice in each group have been done. g – i Ad- Tff2- CTP-Flag reduces splenomegaly ( g , h ) and proportion of CD11b + Gr-1 + cells ( i ) in the spleen of Tff2 -null mice, unpaired t -test, * p < 0.05. One experiment with three mice in each group has been done. j Administration of Ad- Tff2 -CTP-Flag decreases granulocytes/macrophages colonies-forming units in the spleen of wild-type mice treated with DSS. Data obtained from mice treated with 2% DSS for 5 consecutive days and sacrificed on day 19, unpaired t -test, * p < 0.05. k Immunostaining for Gr-1 in the red pulp area in spleens of Tff2 -null mice treated DSS water and received Ad-Fc-CTP (upper raw) or Ad- Tff2 -CTP (lower raw), day 19. Bar size is 100 µm

Article Snippet: All blots were blocked in 5% skim milk in Tris-buffered saline (TBS) plus Tween 20 (0.05%) for 1 h. Affinity-purified primary polyclonal rabbit antibodies were produced against the C-terminus of mouse TFF2 (New England Peptide) and used at concentration 1 μg ml −1 .

Techniques: Injection, Immunostaining

Journal: Cancer Gene Therapy

Article Title: Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer

doi: 10.1038/s41417-018-0036-z

Figure Lengend Snippet: Validation of Ad- Tff2 -CTP-Flag activity in AOM/DSS-induced colon cancer model. a , b Ad- Tff2 suppresses colon tumorigenesis in wild-type mice treated with AOM/DSS. a Colon appearance (left), tumor number (right), b number of splenic CD11b + Gr-1 + cells. Two experiments have been done with 3–4 mice in each group. c , d Ad Tff2 -CTP-Flag suppresses colon tumorigenesis in AOM/DSS-induced colon cancer model. c Colon appearance and tumor number, d proportion CD11b + Gr-1 + cells in the spleen from wild-type mice treated with Ad- Tff2 -CTP versus Ad-Fc-CTP, unpaired t -test, * p < 0.05. Two experiments have been done with 3–4 mice in each group. e Overlapping staining for Flag and Gr-1 in the spleen of Tff2 -null mice injected with Ad- Tff2 -CTP-Flag. Mice were given 2% DSS for 5 consecutive days, then seven days later adenovirus was administrated via tail vein and mice were sacrificed in one week after adenovirus administration. Bar size is 50 µm. f BrdU incorporation in splenic CD11b + Gr-1 + cells of wild-type mice injected with Ad-Fc-CTP-Flag compare with Ad- Tff2 -CTP-Flag, unpaired t-test. Wild-type mice were treated 2% DSS for 5 consecutive days, then on day 7 post treatment with DSS single injection of Ad- Tff2 -CTP or Ad-Fc-CTP-Flag (both 5 × 10 8 pfu) was administered via tail vein injection. Mice were sacrificed on day 19 after start of DSS treatment, BrdU was injected three hours before sacrifice

Article Snippet: All blots were blocked in 5% skim milk in Tris-buffered saline (TBS) plus Tween 20 (0.05%) for 1 h. Affinity-purified primary polyclonal rabbit antibodies were produced against the C-terminus of mouse TFF2 (New England Peptide) and used at concentration 1 μg ml −1 .

Techniques: Biomarker Discovery, Activity Assay, Staining, Injection, BrdU Incorporation Assay

Journal: Cancer Gene Therapy

Article Title: Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer

doi: 10.1038/s41417-018-0036-z

Figure Lengend Snippet: Clearance of recombinant mouse TFF2 and fusion TFF2-CTP-Flag from the blood. a – c Tff2- null mice were tail vein injected with equal molar amount of purified recombinant TFF2 and TFF2-CTP-Flag per kg of mouse weight. At indicated time points blood was taken and assayed for TFF2 and TFF2-CTP-Flag by western blot with antibodies produced against C-terminus of TFF2 molecule ( a , b ) and ELISA ( c ). Excretion of TFF2 with the urine was analyzed by western blot ( a ). ELISA data are plotted as a mean of value and standard deviation at each time point. d Dose-dependent downregulation of CCND1 mRNA in CD11b + Gr-1 + cells upon administration of recombinant wild-type TFF2 and fusion TFF2-CTP-Flag protein. Tff2 -null mice were given 2.5% DSS for 5 days, then CD11b + Gr-1 + were sorted and cultured with recombinant TFF2 and TFF2-CTP-Flag in indicated concentrations for a 7 days

Article Snippet: All blots were blocked in 5% skim milk in Tris-buffered saline (TBS) plus Tween 20 (0.05%) for 1 h. Affinity-purified primary polyclonal rabbit antibodies were produced against the C-terminus of mouse TFF2 (New England Peptide) and used at concentration 1 μg ml −1 .

Techniques: Recombinant, Injection, Purification, Western Blot, Produced, Enzyme-linked Immunosorbent Assay, Standard Deviation, Cell Culture