Journal: World Journal of Gastrointestinal Oncology

Article Title: Taurine suppresses gastric intestinal metaplasia in patient-derived organoids and Atp4a -/- mice

doi: 10.4251/wjgo.v18.i2.114161

Figure Lengend Snippet: Taurine increases the mucin 5AC (MUC5AC) level and inhibits the expression of caudal type homeobox 2, MUC2, and Trefoil factor family 3 in Atp4a -/- mice. A: Expression of caudal type homeobox 2 (CDX2), mucin 2 (MUC2), Trefoil factor family 3 (TFF3) and MUC5AC in Atp4a -/- mouse gastric tissues was assessed via immunohistochemistry (IHC) staining ( n = 6); B: IHC scores of CDX2, MUC2, and TFF3 ( n = 6); C: IHC score of MUC5AC ( n = 6). a P < 0.01 vs Atp4a -/- ; b P < 0.01 vs wild-type (WT).

Article Snippet: The gastric tissue sections were dewaxed, rehydrated, and incubated with primary antibodies against MUC5AC (1:200, GTX11335; GeneTex, Irvine, CA, United States), CDX2 (1:100, ab101532; Abcam, Cambridge, United Kingdom), MUC2 (1:2000, 27675-1-AP; Proteintech, Wuhan, China), and TFF3 (1:800, 23277-1-AP; Proteintech).

Techniques: Expressing, Immunohistochemistry

Journal: World Journal of Gastrointestinal Oncology

Article Title: Taurine suppresses gastric intestinal metaplasia in patient-derived organoids and Atp4a -/- mice

doi: 10.4251/wjgo.v18.i2.114161

Figure Lengend Snippet: Taurine downregulates the protein expression of caudal type homeobox 2 and Trefoil factor family 3 in gastric intestinal metaplasia control organoids. A-C: Western blotting was conducted to assess the protein levels of caudal type homeobox 2 (CDX2) and Trefoil factor family 3 (TFF3); D-I: Relative protein levels were quantified. a P < 0.01 vs normal control; b P < 0.05 vs gastric intestinal metaplasia (GIM) control (CON); c P < 0.01 vs GIM CON.

Article Snippet: The gastric tissue sections were dewaxed, rehydrated, and incubated with primary antibodies against MUC5AC (1:200, GTX11335; GeneTex, Irvine, CA, United States), CDX2 (1:100, ab101532; Abcam, Cambridge, United Kingdom), MUC2 (1:2000, 27675-1-AP; Proteintech, Wuhan, China), and TFF3 (1:800, 23277-1-AP; Proteintech).

Techniques: Expressing, Control, Western Blot

Journal: Alcoholism, clinical and experimental research

Article Title: Blood Biomarkers of Intestinal Epithelium Damage Regenerating Islet-derived Protein 3α and Trefoil Factor 3 Are Persistently Elevated in Patients with Alcoholic Hepatitis

doi: 10.1111/acer.14579

Figure Lengend Snippet: (A-C) Scatter plots showing plasma levels of REG3α, TFF3, and I-FABP in AH patients, HDC, and HC at the baseline and 6 month (D180) and 12 month (D360) follow-ups. Kruskal-Wallis test with Dunn’s corrections was performed to compare plasma levels among 3 groups at baseline. Mann Whitney test was used for compare AH versus HDC individuals at 6 and 12 months. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. Horizontal lines represent the median. (D-F) Dot plots showing the correlation between plasma levels of REG3α, TFF3, and I-FABP in AH patients. Spearman’s correlation was used to calculate the association. r, coefficient. AH, alcoholic hepatitis; HDC, heavy drinking control; HC, healthy control.

Article Snippet: ELISA and Multiplex Immunoassays Levels of REG3α, TFF3, I-FABP (intestinal fatty acid binding protein), and IL-22 in plasma samples were quantified using the Human REG3α DuoSet ELISA Kit, Human TFF3 DuoSet ELISA kit, Human I-FABP DuoSet ELISA kit, and Human IL-22 Quantikine ELISA kit (R&D Systems, Minneapolis, MN), respectively, according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, MANN-WHITNEY, Control

Journal: Alcoholism, clinical and experimental research

Article Title: Blood Biomarkers of Intestinal Epithelium Damage Regenerating Islet-derived Protein 3α and Trefoil Factor 3 Are Persistently Elevated in Patients with Alcoholic Hepatitis

doi: 10.1111/acer.14579

Figure Lengend Snippet: Scatter plots showing the levels of REG3α (A) and TFF3 (B) at 6 month (D180) and 12 month (D360) follow-ups in abstinent AH patients and HDC. Mann Whitney test comparing AH vs HDC at 6 and 12 months. The longitudinal graphs showing changes of the plasma levels of REG3α (C) and TFF3 (D) in AH patients and HDC. Friedman rank sum test with Dunn’s corrections was used to compare these the differences between day 0 and 6 or 12 months (n=10 for AH and n=11 for HDC). *p < 0.05; ***p < 0.001; ns, not significant; horizontal lines represent the median; AH, alcoholic hepatitis; HDC, heavy drinking control.

Article Snippet: ELISA and Multiplex Immunoassays Levels of REG3α, TFF3, I-FABP (intestinal fatty acid binding protein), and IL-22 in plasma samples were quantified using the Human REG3α DuoSet ELISA Kit, Human TFF3 DuoSet ELISA kit, Human I-FABP DuoSet ELISA kit, and Human IL-22 Quantikine ELISA kit (R&D Systems, Minneapolis, MN), respectively, according to the manufacturer’s instructions.

Techniques: MANN-WHITNEY, Clinical Proteomics, Control

Journal: Alcoholism, clinical and experimental research

Article Title: Blood Biomarkers of Intestinal Epithelium Damage Regenerating Islet-derived Protein 3α and Trefoil Factor 3 Are Persistently Elevated in Patients with Alcoholic Hepatitis

doi: 10.1111/acer.14579

Figure Lengend Snippet: Correlations of REG3α, TFF3, I-FABP, and IL-22 with clinical parameters in AH patients

Article Snippet: ELISA and Multiplex Immunoassays Levels of REG3α, TFF3, I-FABP (intestinal fatty acid binding protein), and IL-22 in plasma samples were quantified using the Human REG3α DuoSet ELISA Kit, Human TFF3 DuoSet ELISA kit, Human I-FABP DuoSet ELISA kit, and Human IL-22 Quantikine ELISA kit (R&D Systems, Minneapolis, MN), respectively, according to the manufacturer’s instructions.

Techniques:

Journal: Alcoholism, clinical and experimental research

Article Title: Blood Biomarkers of Intestinal Epithelium Damage Regenerating Islet-derived Protein 3α and Trefoil Factor 3 Are Persistently Elevated in Patients with Alcoholic Hepatitis

doi: 10.1111/acer.14579

Figure Lengend Snippet: (A-B) Scatter plots comparing plasma levels of REG3α (A) and TFF3 (B) in survivors and deceased AH patients. Mann Whitney test was used for compare the difference. **p < 0.01. ns, not significant. Horizontal lines represent the median. (C-D) Kaplan-Meier curves showing 30 day survival according to baseline levels of REG3α (C) and TFF3 (D) in AH patients. The median concentration was used as the cut-off to define patients with low or high concentration. Log-rank test was used for the analysis.*p < 0.05. AH, alcoholic hepatitis.

Article Snippet: ELISA and Multiplex Immunoassays Levels of REG3α, TFF3, I-FABP (intestinal fatty acid binding protein), and IL-22 in plasma samples were quantified using the Human REG3α DuoSet ELISA Kit, Human TFF3 DuoSet ELISA kit, Human I-FABP DuoSet ELISA kit, and Human IL-22 Quantikine ELISA kit (R&D Systems, Minneapolis, MN), respectively, according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, MANN-WHITNEY, Concentration Assay

Journal: Alcoholism, clinical and experimental research

Article Title: Blood Biomarkers of Intestinal Epithelium Damage Regenerating Islet-derived Protein 3α and Trefoil Factor 3 Are Persistently Elevated in Patients with Alcoholic Hepatitis

doi: 10.1111/acer.14579

Figure Lengend Snippet: Correlations of REG3α, TFF3, I-FABP, and IL-22 with BT markers and inflammatory/growth factors in AH patients

Article Snippet: ELISA and Multiplex Immunoassays Levels of REG3α, TFF3, I-FABP (intestinal fatty acid binding protein), and IL-22 in plasma samples were quantified using the Human REG3α DuoSet ELISA Kit, Human TFF3 DuoSet ELISA kit, Human I-FABP DuoSet ELISA kit, and Human IL-22 Quantikine ELISA kit (R&D Systems, Minneapolis, MN), respectively, according to the manufacturer’s instructions.

Techniques:

Journal: Mucosal immunology

Article Title: A Trefoil factor 3-Lingo2 axis restrains proliferative expansion of type-1 T helper cells during GI nematode infection

doi: 10.1016/j.mucimm.2024.02.003

Figure Lengend Snippet: (A) TFF3 protein levels in the sera of WT mice by ELISA. Data mean +/− SEM, 3–7, mice/time point, statistical comparison by one way ANOVA. (B) Intestinal worm burden and (C) fecal egg numbers, shown as log10 eggs per gram of feces in WT vs Tff3KO mice at 35 days post infection. (D) Representative flow plots of activated/effector IFNγ+ MLN cells from infected WT and Tff3KO before and after in vitro stimulated with PMA and ionomycin. (E) Percentage of TCR-β+CD4+CD44+cells that are IFNγ+. (F-I) mRNA expression levels of (F) interferon gamma (IFNγ), (G) interleukin 5 (IL-5,) (H) Retnlb (RELM-β), and (I) Mucin 2 (MUC2) (all normalized to Gapdh) in the cecum of infected WT and Tff3KO. Data are means +/− SEM, representatives of three independent experiments, 4–5 (parasitology in B and C) or 4–7 (for D-I) mice/group respectively, all statistical comparisons are unpaired t tests.

Article Snippet: TFF3 sera concentrations were determined using commercially available mouse TFF3 ELISA kit (Novus Biologicals NP3–08180).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Infection, In Vitro, Expressing

Journal: Mucosal immunology

Article Title: A Trefoil factor 3-Lingo2 axis restrains proliferative expansion of type-1 T helper cells during GI nematode infection

doi: 10.1016/j.mucimm.2024.02.003

Figure Lengend Snippet: (A) Schematic of anti-IL-12 IgG2a antibody or isotype control treatment following T. muris infection. (B) Fecal eggs and (C) worm burden at 35dpi by genotype and antibody treatment. (D-F) Secretion of (D) IL-5, (D) IL-13, and (F) IFNγ by MLN cells isolated from treated CD4creLingo2fl/fl mice at 35dpi and stimulated ex-vivo with anti-CD3/CD28 mAb, measured by ELISA. (G-L) Representative flow plot and percentage of CD4+ T cells expressing (G, H) CD44 and GATA3, (I, J) CD44 and Tbet, (K, L) Tbet and IFNγ. Data are mean ± SEM, 3–9 mice/group, statistical analysis by unpaired t-tests, and representative of two experiments.

Article Snippet: TFF3 sera concentrations were determined using commercially available mouse TFF3 ELISA kit (Novus Biologicals NP3–08180).

Techniques: Control, Infection, Isolation, Ex Vivo, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Mucosal immunology

Article Title: A Trefoil factor 3-Lingo2 axis restrains proliferative expansion of type-1 T helper cells during GI nematode infection

doi: 10.1016/j.mucimm.2024.02.003

Figure Lengend Snippet: (A) Naïve CD4+ T cells were harvested from WT mice and TCR activated with anti-CD3/CD28 antibodies and cultured under TH0 (anti-CD3/CD28 mAb TCR activation only), or TH1 polarizing conditions either with or without TFF3-Fc treatment. Cells were then rested and expanded with recombinant IL-2 and analyzed by flow cytometry. (B) Representative flow plots of Ki-67+ CD4+ T cells with graph quantifying percentage of TCR-β+CD4+CD44+cells that are Ki-67+. (C) Histograms and graph showing quantification of median fluorescence intensity (MFI) of Ki-67+ staining of TFF3-Fc treated and untreated TH0 and TH1 polarized cells. (D-E) Representative flow plots and graphical percentages of (D) Tbet+ (E) Tbet+ Ki-67+ in TH1 polarized cells with and without TFF3-Fc treatment. (F) T cells were isolated from WT or Lingo2KO mice activated and cultured under TH0, TH1 (not shown, same as A), TH2, TH17 and TREG polarizing conditions and Lingo2 gene expression was assessed by qRT-PCR (normalized to Gapdh). Fold change (2 −ΔΔCt ) in Lingo2 mRNA expression in (G) activated (TH0) WT and Lingo2KO CD4+ T cells relative to unstimulated controls (H) and under TH1, TH2, TH17 and TREG polarizing conditions. Cultures derived from naïve CD4+ cells isolated from 3–4 mice/group and kept separate as biological and technical replicas. Data are mean +/− SEM, representative of two independent experiments. Significance determined by unpaired t-test, one way or two-way ANOVA.

Article Snippet: TFF3 sera concentrations were determined using commercially available mouse TFF3 ELISA kit (Novus Biologicals NP3–08180).

Techniques: Cell Culture, Activation Assay, Recombinant, Flow Cytometry, Fluorescence, Staining, Isolation, Gene Expression, Quantitative RT-PCR, Expressing, Derivative Assay

Journal: Mucosal immunology

Article Title: A Trefoil factor 3-Lingo2 axis restrains proliferative expansion of type-1 T helper cells during GI nematode infection

doi: 10.1016/j.mucimm.2024.02.003

Figure Lengend Snippet: (A) Fecal egg numbers, shown as log10 eggs per gram of feces and worm burden of WT versus Lingo2KO mice 35 days post infection. (B) Fecal egg numbers, shown as log10 eggs per gram of feces and worm burden in control (CD4cre and Lingo2fl/fl) versus CD4creLingo2fl/fl mice at 35 days post infection. (C) Representative images of H&E and PAS stained cecal tissue from infected Lingo2fl/fl versus CD4creLingo2fl/fl. (D) Quantification of goblet cells from PAS stained sections from Lingo2fl/fl versus CD4creLingo2fl/fl (E-G) mRNA expression levels of (E) trefoil factor 3 (TFF3) (F) Retnlb (RELM-β) (G) Mucin 2 (MUC2) (all normalized to Gapdh) in the cecum of infected CD4cre and CD4cre Lingo2fl/fl mice. Data are means +/− SEM, representative of 2–3 independent experiments (A) 4, (B,D) 7–10, or (E-G) 8–10 mice/group respectively, unpaired t test or one-way ANOVA shown.

Article Snippet: TFF3 sera concentrations were determined using commercially available mouse TFF3 ELISA kit (Novus Biologicals NP3–08180).

Techniques: Infection, Control, Staining, Expressing

Journal: British Journal of Nutrition

Article Title: Phytic acid improves intestinal mucosal barrier damage and reduces serum levels of proinflammatory cytokines in a 1,2-dimethylhydrazine-induced rat colorectal cancer model

doi: 10.1017/s0007114518001290

Figure Lengend Snippet: Fig. 4. Real-time PCR analysis of mucin 2 (MUC2) (a) and trefoil factor 3 (TFF3) (b) mRNA expression levels. The normal colon tissues were selected in the control group (CG) and the colon tumour tissues were selected in the other groups. β-Actin was used as a loading control. MG, model group; PA, phytic acid; LPG, low-PA-dose group; MPG, middle-PA-dose group; HPG, high-PA-dose group. **P<0·01, CG compared with the MG; †† P< 0·01, PA treatment groups compared with the MG.

Article Snippet: The following antibodies were used in the Western blot analysis: rabbit anti-MUC2 (Abcam), claudin-1 (Novus), TFF3 (Novus) and E-cadherin (Danvers).

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

Journal: British Journal of Nutrition

Article Title: Phytic acid improves intestinal mucosal barrier damage and reduces serum levels of proinflammatory cytokines in a 1,2-dimethylhydrazine-induced rat colorectal cancer model

doi: 10.1017/s0007114518001290

Figure Lengend Snippet: Fig. 6. Western blot analysis of mucin 2 (MUC2) and trefoil factor 3 (TFF3) protein expression. The normal colon tissues were selected in the control group (CG) and the colon tumour tissues were selected in the other groups. β-Actin was used as a loading control. (a) Representative blots from one of three separate experiments; (b) relative band intensities of MUC2 based on densitometry; (c) relative band intensities of TFF3 based on densitometry. MG, model group; PA, phytic acid; LPG, low-PA-dose group; MPG, middle-PA- dose group; HPG, high-PA-dose group. ** P < 0·01, CG compared with the MG; †† P < 0·01, PA treatment groups compared with the MG.

Article Snippet: The following antibodies were used in the Western blot analysis: rabbit anti-MUC2 (Abcam), claudin-1 (Novus), TFF3 (Novus) and E-cadherin (Danvers).

Techniques: Western Blot, Expressing, Control

Journal: Biomarkers

Article Title: The Diagnostic Value of Serum Trefoil Factor 3 and Pepsinogen combination in Chronic Atrophic Gastritis: A Retrospective Study Based on a Gastric Cancer Screening Cohort in the Community Population

doi: 10.1080/1354750x.2024.2400927

Figure Lengend Snippet: Figure 1. Study population flowchart. A total of 2360 patients with chronic gastritis were screened according to exclusion criteria, resulting in 1609 patients with chronic gastritis. Further research included 540 patients who underwent anti-H. Pylori antibody, PG, TFF3 testing, gastroscopy, and histopathological examination. Among them, there were 385 patients in the test set (CAG = 225, CSG = 160) and 155 patients in the validation set (CAG = 106, CSG = 49).

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) for serum TFF3 serum samples stored in a −80 °c refrigerator were taken out, centrifuged at 1000 g for 20 min, and the supernatant was diluted by 1:15. human tFF3 elisa kits (elabscience, Wuhan) were used to determine serum tFF3 levels. the specific steps are as follows: a layer of human-specific tFF3 monoclonal antibody was pre-coated on the microplate. then, 100 μl of standards and samples were transferred to the wells, incubated at 37 °c for 90 min, the liquid in the wells was discarded, 100 μl of biotinylated antibody/antigen working solution was added to each well, incubated at 37 °c for 60 min, washed three times, 350 μl of washing solution was added to each well, 100 μl of human tFF3 polyclonal antibody was added to each well, and incubated at 37 °c for 30 min. the plate was washed five times, 90 μl of substrate solution was added to each well, incubated at 37 °c in the dark for 15 min, until a clear color gradient appears in the standard well.

Techniques: Biomarker Discovery

Journal: Biomarkers

Article Title: The Diagnostic Value of Serum Trefoil Factor 3 and Pepsinogen combination in Chronic Atrophic Gastritis: A Retrospective Study Based on a Gastric Cancer Screening Cohort in the Community Population

doi: 10.1080/1354750x.2024.2400927

Figure Lengend Snippet: Figure 2. Scatter plot of expression of different serum biomarkers in the CAG and CSG group. The expression of serum PG1(a), PGII (b), PGI/PGII (c), and TFF3(d) were measured. 540 patients were divided into two groups, with 209 patients for chronic superficial gastritis (CSG) and 331 patients for chronic atrophic gastritis (CAG). The horizontal axis represents different groupings, and the vertical axis represents the expression levels of different markers. P < 0.05 indicates statistical significance.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) for serum TFF3 serum samples stored in a −80 °c refrigerator were taken out, centrifuged at 1000 g for 20 min, and the supernatant was diluted by 1:15. human tFF3 elisa kits (elabscience, Wuhan) were used to determine serum tFF3 levels. the specific steps are as follows: a layer of human-specific tFF3 monoclonal antibody was pre-coated on the microplate. then, 100 μl of standards and samples were transferred to the wells, incubated at 37 °c for 90 min, the liquid in the wells was discarded, 100 μl of biotinylated antibody/antigen working solution was added to each well, incubated at 37 °c for 60 min, washed three times, 350 μl of washing solution was added to each well, 100 μl of human tFF3 polyclonal antibody was added to each well, and incubated at 37 °c for 30 min. the plate was washed five times, 90 μl of substrate solution was added to each well, incubated at 37 °c in the dark for 15 min, until a clear color gradient appears in the standard well.

Techniques: Expressing

Journal: Biomarkers

Article Title: The Diagnostic Value of Serum Trefoil Factor 3 and Pepsinogen combination in Chronic Atrophic Gastritis: A Retrospective Study Based on a Gastric Cancer Screening Cohort in the Community Population

doi: 10.1080/1354750x.2024.2400927

Figure Lengend Snippet: Figure 4. Expression of different serum biomarkers in H. pylori-positive/negative CAG. To analyze the expression levels of PG1(a), PGII (B), PGI/PGII (C), and TFF3 (D) in serum under different H. Pylori infection status. The x-axis represents different disease groups, and the y-axis represents the expression levels of the different biomarkers. H. Pylori positive is represented by black bars, while H. Pylori negative is represented by red bars. P < 0.05 is considered statistically significant.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) for serum TFF3 serum samples stored in a −80 °c refrigerator were taken out, centrifuged at 1000 g for 20 min, and the supernatant was diluted by 1:15. human tFF3 elisa kits (elabscience, Wuhan) were used to determine serum tFF3 levels. the specific steps are as follows: a layer of human-specific tFF3 monoclonal antibody was pre-coated on the microplate. then, 100 μl of standards and samples were transferred to the wells, incubated at 37 °c for 90 min, the liquid in the wells was discarded, 100 μl of biotinylated antibody/antigen working solution was added to each well, incubated at 37 °c for 60 min, washed three times, 350 μl of washing solution was added to each well, 100 μl of human tFF3 polyclonal antibody was added to each well, and incubated at 37 °c for 30 min. the plate was washed five times, 90 μl of substrate solution was added to each well, incubated at 37 °c in the dark for 15 min, until a clear color gradient appears in the standard well.

Techniques: Expressing, Infection

Journal: Cancers

Article Title: Inhibition of TFF3 Enhances Sensitivity—and Overcomes Acquired Resistance—to Doxorubicin in Estrogen Receptor-Positive Mammary Carcinoma

doi: 10.3390/cancers11101528

Figure Lengend Snippet: Inhibition of TFF3 enhances cellular sensitivity to doxorubicin. ( a – b ). MCF-7, ZR-75-1 and BT-474 cells ( a ) pre-incubated with 20nM of two TFF3 siRNA combined overnight or ( b ) treated with 10 μM AMPC were treated with increasing doses of doxorubicin for 72 h in monolayer culture. Cell viability was quantified using the AlamarBlue cell viability assay where the 50% inhibitory concentration (IC 50 ) values for doxorubicin were determined using GraphPad Prism 5. Western blot analysis determined the protein expression of TFF3. β-ACTIN was used as input control. Band intensities were quantified by ImageJ and normalized to input control, where intensity ratio of scrambled siRNA/vehicle DMSO treatment was set to 1. ( c – e ). MCF-7, ZR-75-1 and BT-474 cells were treated with doxorubicin with or without AMPC ( c ) over a period of 6 days in monolayer culture (MCF-7: 50 nM dox, 1 μM AMPC; ZR-75-1: 200 nM dox, 5 μM AMPC; BT-474: 100 nM dox, 5 μM AMPC); ( d ) in monolayer culture at low cell density until foci formation (MCF-7: 50 nM dox, 1 μM AMPC; ZR-75-1: 25 nM dox, 2 μM AMPC; BT-474: 10 nM dox, 1 μM AMPC); and ( e ) for 9 days after 5 days pre-culture in medium containing 5% FBS and 4% Matrigel (MCF-7: 50 nM dox, 2 μM AMPC; ZR-75-1: 200 nM dox, 5 μM AMPC; BT-474: 50 nM dox, 5 μM AMPC). Cell viability was quantified using the AlamarBlue cell viability assay. Bar charts show means ± standard deviations. * denotes doxorubicin vs combination treatment; ◆ denotes AMPC vs combination treatment where * P < 0.05, ** P < 0.01 and *** P < 0.001 (Student’s t -test).

Article Snippet: The TFF3 levels in the serum were quantified through colorimetric determination using a specific human TFF3 Quantikine ELISA Kit (DTFF30, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Inhibition, Incubation, Viability Assay, Concentration Assay, Western Blot, Expressing, Control

Journal: Cancers

Article Title: Inhibition of TFF3 Enhances Sensitivity—and Overcomes Acquired Resistance—to Doxorubicin in Estrogen Receptor-Positive Mammary Carcinoma

doi: 10.3390/cancers11101528

Figure Lengend Snippet: AMPC inhibits doxorubicin-induced AKT activation and enhances doxorubicin-induced apoptosis in ER+MC cells. MCF-7 cells were treated with 500nM doxorubicin with or without 10 μM AMPC in monolayer culture. ( a – b ). Western blot analysis determined the protein expression of ( a ) TFF3, AKT and ( b ) apoptotic-related proteins after drug treatment for 24 h. β-ACTIN was used as input control. Band intensities were quantified by ImageJ and normalized to input control/total protein for phosphorylated proteins, where intensity ratio of vehicle DMSO treatment was set to 1. ( c ) MCF-7 cells were stained with Annexin V-PI stain after drug treatment for 48 h. Percentage (%) apoptotic cells were analyzed by flow cytometry. Bar charts show means ± standard deviations. * P < 0.05, ** P < 0.01 and *** P < 0.001 (Student’s t -test).

Article Snippet: The TFF3 levels in the serum were quantified through colorimetric determination using a specific human TFF3 Quantikine ELISA Kit (DTFF30, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Activation Assay, Western Blot, Expressing, Control, Staining, Flow Cytometry

Journal: Cancers

Article Title: Inhibition of TFF3 Enhances Sensitivity—and Overcomes Acquired Resistance—to Doxorubicin in Estrogen Receptor-Positive Mammary Carcinoma

doi: 10.3390/cancers11101528

Figure Lengend Snippet: Doxorubicin resistant ER+MC cells exhibit elevated levels of TFF3. ( a – b ) Baseline levels of TFF3 ( a ) mRNA and ( b ) protein levels were analyzed by qPCR and western blot analysis respectively in control and doxorubicin resistant MCF-7 cells. Band intensities were quantified by ImageJ and normalized to input control, where the intensity ratio of doxorubicin control cells was set to 1. Bar charts show means ± standard deviations. * P < 0.05, ** P < 0.01 and *** P < 0.001 (Student’s t-test).

Article Snippet: The TFF3 levels in the serum were quantified through colorimetric determination using a specific human TFF3 Quantikine ELISA Kit (DTFF30, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Western Blot, Control

Journal: Cancers

Article Title: Inhibition of TFF3 Enhances Sensitivity—and Overcomes Acquired Resistance—to Doxorubicin in Estrogen Receptor-Positive Mammary Carcinoma

doi: 10.3390/cancers11101528

Figure Lengend Snippet: TFF3 inhibition re-sensitizes doxorubicin resistant ER+MC cells to doxorubicin-induced apoptosis. Control and doxorubicin resistant MCF-7 cells ( a ) pre-incubated with 20 nM of two TFF3 siRNA combined or ( b ) co-treated with 1 μM AMPC were treated with increasing doses of doxorubicin for 72 h in monolayer culture. ( c ) Increasing doses of doxorubicin and AMPC were treated to control and doxorubicin resistant MCF-7 cells at a fixed ratio of 1:20 for 72 h in monolayer culture. Combination index (CI) and dose reduction index (DRI) were tabulated using CalcuSyn software by Chou-Talalay . CI 50–80 and DRI 50–80 denotes average combination index and average dose reduction index respectively at 50–80% cell death. CI > 1 indicates antagonism; CI = 1 indicates an additive effect; and CI < 1 indicates synergism. DRI > 1 is favorable dose reduction that leads to toxicity reduction. ( d – f ). Control and doxorubicin resistant ER+MC cells were treated with doxorubicin with or without AMPC ( d ) for 3 days in monolayer culture (50 nM dox, 1 μM AMPC); ( e ) in monolayer culture at low cell density until foci formation (25 nM dox, 2 μM AMPC); and ( f ) for 9 days after 5 days pre-culture in medium containing 5% FBS and 4% Matrigel (100 nM dox, 5 μM AMPC). ( g ) Western blot analysis for protein levels of TFF3, AKT and BAD after 10 μM AMPC treatment for 24 h. C denotes control cells while R denotes doxorubicin resistant ER+MC cells. β-ACTIN was used as input control. Band intensities were quantified by ImageJ and normalized to input control/total proteins for phosphorylated proteins, where intensity ratio of control cells treated with vehicle DMSO was set to 1. ( h – i ). Total apoptosis was analyzed in the control and doxorubicin resistant MCF-7 cells treated with combination of doxorubicin with or without AMPC for ( h ) 48 h, followed by Annexin V-PI staining and quantification by flow cytometry; or ( i ) 24 h, followed by TUNEL staining and visualization by fluorescent microscopy. % of TUNEL positive cells was quantified by ImageJ. Cell viability was quantified using the AlamarBlue cell viability assay and 50% inhibitory concentration (IC 50 ) values for doxorubicin were determined using GraphPad Prism 5. The scale bar represents 50 μm. Bar charts show means ± standard deviations. * P < 0.05, ** P < 0.01 and *** P < 0.001 (Student’s t -test).

Article Snippet: The TFF3 levels in the serum were quantified through colorimetric determination using a specific human TFF3 Quantikine ELISA Kit (DTFF30, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Inhibition, Control, Incubation, Software, Western Blot, Staining, Flow Cytometry, TUNEL Assay, Microscopy, Viability Assay, Concentration Assay

Journal: Cancers

Article Title: Inhibition of TFF3 Enhances Sensitivity—and Overcomes Acquired Resistance—to Doxorubicin in Estrogen Receptor-Positive Mammary Carcinoma

doi: 10.3390/cancers11101528

Figure Lengend Snippet: Inhibition of TFF3 by AMPC inhibits the in vivo growth of ER+ MC xenografts. Doxorubicin control and resistant ER+ MCF-7 cells were injected orthotopically into the mammary fat pad of nude mice. ( a ) Tumour volumes and ( b ) % change in tumour volume in mice were recorded daily with vehicle or AMPC treatment over 15 days. ( c ) Representative photographs of tumour sites (left) and the corresponding micrographs of the H&E-stained tumour sections at 200× magnification (right). ( d ) Representative photographs of tumours from each treatment groups and the average tumour weights. ( e – h ) Immunohistochemistry (IHC) staining was performed on tumours for Ki67, TUNEL and TFF3. Representative micrographs were taken at 200x magnification and staining intensity were scored using the immunoreactivity scoring (IRS) system. ( i ) Serum levels of TFF3 was determined by ELISA. Bar charts represents the mean ± SD of the averages of 6-8 mice scored. Scale bars represents 20 μm. Results were statistically analysed with one-way ANOVA followed by Tukey’s post hoc test. Statistical significance, * P < 0.05, ** P < 0.01 and *** P < 0.001, compared with the vehicle-treated group.

Article Snippet: The TFF3 levels in the serum were quantified through colorimetric determination using a specific human TFF3 Quantikine ELISA Kit (DTFF30, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Inhibition, In Vivo, Control, Injection, Staining, Immunohistochemistry, TUNEL Assay, Enzyme-linked Immunosorbent Assay

Journal: Biological Procedures Online

Article Title: Production of Monoclonal Antibodies against Human Trefoil Factor 3 and Development of a Modified-Sandwich ELISA for Detection of Trefoil Factor 3 Homodimer in Saliva

doi: 10.1186/s12575-017-0064-3

Figure Lengend Snippet: Antibody responses of immunized mouse and characterization of anti-TFF3 monoclonal antibodies. a Mouse was immunized with recombinant TFF3 three times at two-week intervals. Blood was collected before (pre-immunization: square marker and gray line) and 7 days after the third immunization (post-immunization: triangle marker and black line). Various dilutions (as indicated) of the mouse sera were investigated for the presence of anti-TFF3 antibodies by indirect ELISA. Values are presented as mean ± SD of two independent experiments. Paired, two tailed Student’s t test was used to assess significance level of anti-TFF3 antibody in pre-and post-immunization sera ( P < 0.01). b Indirect ELISA was performed for the determination of the specificity of the generated anti-TFF3 mAbs (TFF-116, TFF-286 and TFF-298). Human recombinant (r) TFF1, TFF2 and TFF3 or ( c ) human recombinant monomeric and dimeric TFF3 were coated on an ELISA plate. Commercial anti-TFF1, anti-TFF2 and anti-TFF3 mAbs were used as positive controls for reacting with their corresponding antigens. IgG1,κ was used as the isotype-matched control mAb (isotype control). Bar graphs represent mean ± SD of two independent experiments. There was statistically significantly higher reactivity in tested mAb compared with isotype matched control mAb (all P -values <0.05)

Article Snippet: The plates were blocked with 2% skimmed milk in PBS at 37 °C for 1 h. Mouse sera, hybridoma culture supernatants, or commercial antibodies, including anti-TFF1 mAb (Sigma-Aldrich), anti-TFF2 mAb (R&D Systems, Minnesota, USA) and anti-TFF3 mAb (R&D Systems), were added and incubated at 37 °C for 1 h. The plates were washed and HRP-conjugated rabbit anti-mouse immunoglobulin antibodies (DAKO) were added; thereafter, the plates were incubated at 37 °C for 1 h. Subsequently, 3, 3′, 5, 5′-tetramethylbenzidine (TMB) substrate (Invitrogen, Camarillo, CA, USA) was added.

Techniques: Bioprocessing, Recombinant, Marker, Indirect ELISA, Two Tailed Test, Generated, Enzyme-linked Immunosorbent Assay, Control

Journal: Biological Procedures Online

Article Title: Production of Monoclonal Antibodies against Human Trefoil Factor 3 and Development of a Modified-Sandwich ELISA for Detection of Trefoil Factor 3 Homodimer in Saliva

doi: 10.1186/s12575-017-0064-3

Figure Lengend Snippet: Western blot analysis of anti-human TFF3 monoclonal antibodies. a The Western blotting results were demonstrated with the indicated anti-TFF3 mAbs using human recombinant monomeric TFF3 under reducing conditions. b SDS-PAGE demonstrated the molecular size of human recombinant monomeric TFF3 (mTFF3) and dimeric TFF3 (dTFF3) in non-reducing (NR) and reducing (R) conditions. The proteins were stained using PageBlue™ Protein Staining Solution. The molecular markers (kDa) are indicated on the left. The data was representative of two independent experiments

Article Snippet: The plates were blocked with 2% skimmed milk in PBS at 37 °C for 1 h. Mouse sera, hybridoma culture supernatants, or commercial antibodies, including anti-TFF1 mAb (Sigma-Aldrich), anti-TFF2 mAb (R&D Systems, Minnesota, USA) and anti-TFF3 mAb (R&D Systems), were added and incubated at 37 °C for 1 h. The plates were washed and HRP-conjugated rabbit anti-mouse immunoglobulin antibodies (DAKO) were added; thereafter, the plates were incubated at 37 °C for 1 h. Subsequently, 3, 3′, 5, 5′-tetramethylbenzidine (TMB) substrate (Invitrogen, Camarillo, CA, USA) was added.

Techniques: Western Blot, Bioprocessing, Recombinant, SDS Page, Staining

Journal: Biological Procedures Online

Article Title: Production of Monoclonal Antibodies against Human Trefoil Factor 3 and Development of a Modified-Sandwich ELISA for Detection of Trefoil Factor 3 Homodimer in Saliva

doi: 10.1186/s12575-017-0064-3

Figure Lengend Snippet: Schematic diagram demonstrating the principle behind the developed sandwich ELISA detecting dimeric TFF3. a The steps of the developed sandwich ELISA: ( a ). First, anti-TFF3 mAb is coated on the ELISA plate. b . Human recombinant TFF3 or saliva is added. The TFF3 present in the sample binds to the mAbs. c . The FITC-conjugated secondary anti-TFF3 mAb (which is the same clone of the coated mAb) is added. d. HRP-conjugated anti-FITC antibody is added and there is a reaction to the FITC-labeled anti-TFF3 mAb. e. Lastly, the TMB substrate is added and the color develops. The intensity of the color is proportional to the amount of the dimeric TFF3 present in the sample. Dimeric TFF3 can bind to both the coated mAb and the secondary mAb and the colorimetric signal can be developed by the reaction of the HRP-conjugated anti-FITC antibody with the TMB substrate. b In contrast, the secondary mAb cannot bind to monomeric TFF3 which cannot develop the colorimetric signal

Article Snippet: The plates were blocked with 2% skimmed milk in PBS at 37 °C for 1 h. Mouse sera, hybridoma culture supernatants, or commercial antibodies, including anti-TFF1 mAb (Sigma-Aldrich), anti-TFF2 mAb (R&D Systems, Minnesota, USA) and anti-TFF3 mAb (R&D Systems), were added and incubated at 37 °C for 1 h. The plates were washed and HRP-conjugated rabbit anti-mouse immunoglobulin antibodies (DAKO) were added; thereafter, the plates were incubated at 37 °C for 1 h. Subsequently, 3, 3′, 5, 5′-tetramethylbenzidine (TMB) substrate (Invitrogen, Camarillo, CA, USA) was added.

Techniques: Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Recombinant, Labeling

Journal: Biological Procedures Online

Article Title: Production of Monoclonal Antibodies against Human Trefoil Factor 3 and Development of a Modified-Sandwich ELISA for Detection of Trefoil Factor 3 Homodimer in Saliva

doi: 10.1186/s12575-017-0064-3

Figure Lengend Snippet: Sandwich ELISA for the measurement of TFF3 homodimer . The sandwich ELISA was developed using a pair of mAbs against TFF3. The three combinations of the capture and detector mAbs, namely TFF-116 and FITC-labeled TFF-116 (116–116 FITC), TFF-286 and FITC-labeled TFF-286 (286–286 FITC) and TFF-298 and FITC-labeled TFF-298 (298–298 FITC), were employed for detecting recombinant TFF3 monomeric and dimeric forms. Bar graphs represent mean ± SD of two independent experiments. There was statistically significantly higher reactivity in dimeric form compared with monomeric and no antigen (all P -values <0.05)

Article Snippet: The plates were blocked with 2% skimmed milk in PBS at 37 °C for 1 h. Mouse sera, hybridoma culture supernatants, or commercial antibodies, including anti-TFF1 mAb (Sigma-Aldrich), anti-TFF2 mAb (R&D Systems, Minnesota, USA) and anti-TFF3 mAb (R&D Systems), were added and incubated at 37 °C for 1 h. The plates were washed and HRP-conjugated rabbit anti-mouse immunoglobulin antibodies (DAKO) were added; thereafter, the plates were incubated at 37 °C for 1 h. Subsequently, 3, 3′, 5, 5′-tetramethylbenzidine (TMB) substrate (Invitrogen, Camarillo, CA, USA) was added.

Techniques: Sandwich ELISA, Labeling, Recombinant

Journal: Biological Procedures Online

Article Title: Production of Monoclonal Antibodies against Human Trefoil Factor 3 and Development of a Modified-Sandwich ELISA for Detection of Trefoil Factor 3 Homodimer in Saliva

doi: 10.1186/s12575-017-0064-3

Figure Lengend Snippet: Detection of human TFF3 homodimer in saliva. a Various concentrations of the dimeric TFF3 were measured by the developed sandwich ELISA using TFF-286 as the capture and detector mAbs. A typical calibration curve covering TFF3 concentrations in the range of 0.5–32 ng/ml was obtained. a representative result from one of five experiment is shown. b Salivary TFF3 concentrations were determined by the developed sandwich ELISA. The levels of the salivary TFF3 concentrations in saliva ( n = 13) are shown. Eight healthy subjects (closed circle) and five oral squamous cell carcinoma (OSCC) patients (open circle) were indicated. The data was representative of two independent experiments

Article Snippet: The plates were blocked with 2% skimmed milk in PBS at 37 °C for 1 h. Mouse sera, hybridoma culture supernatants, or commercial antibodies, including anti-TFF1 mAb (Sigma-Aldrich), anti-TFF2 mAb (R&D Systems, Minnesota, USA) and anti-TFF3 mAb (R&D Systems), were added and incubated at 37 °C for 1 h. The plates were washed and HRP-conjugated rabbit anti-mouse immunoglobulin antibodies (DAKO) were added; thereafter, the plates were incubated at 37 °C for 1 h. Subsequently, 3, 3′, 5, 5′-tetramethylbenzidine (TMB) substrate (Invitrogen, Camarillo, CA, USA) was added.

Techniques: Sandwich ELISA

Journal: Frontiers in Immunology

Article Title: Mode of Proximal Tubule Damage: Differential Cause for the Release of TFF3?

doi: 10.3389/fimmu.2016.00122

Figure Lengend Snippet: Densitometric analysis of proteome array reactivities for TFF3 (A) and characteristic markers of hypoxia (FABP1, VEGF) and inflammation (CXCL16) (B) using cell lysates from HK-2 cells exposed to hypoxia/nutrient starvation (HNS) and replenishment (HNSR) for 24 and 48 h . Densities obtained are shown in (C) . These were normalized to a reference (pos) and the background was subtracted. Data are presented as mean ± SD and were compared using Student’s multiple t -test. p < 0.05 was considered statistically significant. GraphPad Prism 6.07 was used for statistical analysis (GraphPad Software, La Jolla, CA, USA). HIF-1α abundance is increased at the early replenishment time point when normalized to a housekeeping protein β-actin and compared to the normoxic control (D) . Normalized abundance of TFF3 (7 kDa) in cell lysates from HK-2 cells exposed to hypoxia/nutrient starvation (HNS) and replenishment for 24 and 48 h (E) . NDL, normalized density to loading control (Western blot).

Article Snippet: TFF3 was measured by R&D Systems™ Quantikine ® ELISA human TFF3 immunoassay (Minneapolis, MN, USA), and levels were calculated using a linear standard curve.

Techniques: Software, Western Blot

Journal: Frontiers in Immunology

Article Title: Mode of Proximal Tubule Damage: Differential Cause for the Release of TFF3?

doi: 10.3389/fimmu.2016.00122

Figure Lengend Snippet: The human kidney biomarker array (R&D Systems) was used to analyze TFF3 abundance in lysates from cells exposed to immunoglobulin λ light chain (LC) and fatty acid free human serum albumin (FAF-HSA) for 72 h in comparison with control cells (A) . Densities were normalized to a reference (pos), background subtracted and are expressed as ±SD. (B) Abundance of TFF3 (in relation to β-actin) in cell lysates from HK-2 cells exposed 5 mg/ml immunoglobulin λ light chain (LC) or fatty acid-free human serum albumin (FAF-HSA) for 72 h (Western blot). (C) ELISA of TFF3 in the supernatants of HK-2 stimulated with 5 mg/ml LC and FAF-HSA. Untreated cells were used as control. Two independent experiments for the distinct stimulations for 24 and 72 h are shown.

Article Snippet: TFF3 was measured by R&D Systems™ Quantikine ® ELISA human TFF3 immunoassay (Minneapolis, MN, USA), and levels were calculated using a linear standard curve.

Techniques: Biomarker Assay, Comparison, Western Blot, Enzyme-linked Immunosorbent Assay