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Novus Biologicals recombinant tff2

Figure 4. The expression and secretion of <t>trefoil</t> <t>factor</t> <t>2</t> <t>(TFF2)</t> is increased in OGT-deﬁcient hepatocytes. A, the expression of genes that encode secreted proteins in WT and OGT-LKO livers. Top 50 genes are shown based on the ranks of fold change. B–D, mRNA expression of Tff2 (B), Bmp8b (C), and Gpnmb (D) in primary hepatocytes (PH) and liver lysates. Cell and liver lysates were isolated from 4-week-old WT and OGT-LKO mice. n = 3 to 5. E, Western blotting of proteins extracted from WT and KO hepatocyte-conditioned medium. F, immunoﬂuorescence imaging of primary hepatocytes with the antibody against TFF2 (green) and 40,6-diamidino-2-phenylindole (blue). The scale bar represents 25 μm. G and H, mRNA expression of Tff1 and Tff3 in primary hepatocytes and liver lysates. Cell and liver lysates were isolated from 4-week-old WT and OGT-LKO mice. n = 4 to 5. Data are shown as the mean ± SEM. *p < 0.05; **p < 0.01 by unpaired Student’s t test. HSCs, hepatic stellate cells; OGT, O-GlcNAc transferase; OGT-LKO, liver-speciﬁc OGT KO.

Recombinant Tff2, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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recombinant tff2 - by Bioz Stars, 2026-05

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1) Product Images from "Trefoil factor 2 secreted from damaged hepatocytes activates hepatic stellate cells to induce fibrogenesis"

Article Title: Trefoil factor 2 secreted from damaged hepatocytes activates hepatic stellate cells to induce fibrogenesis

Journal: Journal of Biological Chemistry

doi: 10.1016/j.jbc.2021.100887

Figure 4. The expression and secretion of trefoil factor 2 (TFF2) is increased in OGT-deﬁcient hepatocytes. A, the expression of genes that encode secreted proteins in WT and OGT-LKO livers. Top 50 genes are shown based on the ranks of fold change. B–D, mRNA expression of Tff2 (B), Bmp8b (C), and Gpnmb (D) in primary hepatocytes (PH) and liver lysates. Cell and liver lysates were isolated from 4-week-old WT and OGT-LKO mice. n = 3 to 5. E, Western blotting of proteins extracted from WT and KO hepatocyte-conditioned medium. F, immunoﬂuorescence imaging of primary hepatocytes with the antibody against TFF2 (green) and 40,6-diamidino-2-phenylindole (blue). The scale bar represents 25 μm. G and H, mRNA expression of Tff1 and Tff3 in primary hepatocytes and liver lysates. Cell and liver lysates were isolated from 4-week-old WT and OGT-LKO mice. n = 4 to 5. Data are shown as the mean ± SEM. *p < 0.05; **p < 0.01 by unpaired Student’s t test. HSCs, hepatic stellate cells; OGT, O-GlcNAc transferase; OGT-LKO, liver-speciﬁc OGT KO.

Figure Legend Snippet: Figure 4. The expression and secretion of trefoil factor 2 (TFF2) is increased in OGT-deﬁcient hepatocytes. A, the expression of genes that encode secreted proteins in WT and OGT-LKO livers. Top 50 genes are shown based on the ranks of fold change. B–D, mRNA expression of Tff2 (B), Bmp8b (C), and Gpnmb (D) in primary hepatocytes (PH) and liver lysates. Cell and liver lysates were isolated from 4-week-old WT and OGT-LKO mice. n = 3 to 5. E, Western blotting of proteins extracted from WT and KO hepatocyte-conditioned medium. F, immunoﬂuorescence imaging of primary hepatocytes with the antibody against TFF2 (green) and 40,6-diamidino-2-phenylindole (blue). The scale bar represents 25 μm. G and H, mRNA expression of Tff1 and Tff3 in primary hepatocytes and liver lysates. Cell and liver lysates were isolated from 4-week-old WT and OGT-LKO mice. n = 4 to 5. Data are shown as the mean ± SEM. *p < 0.05; **p < 0.01 by unpaired Student’s t test. HSCs, hepatic stellate cells; OGT, O-GlcNAc transferase; OGT-LKO, liver-speciﬁc OGT KO.

Techniques Used: Expressing, Isolation, Western Blot, Imaging

Figure 5. TFF2 promotes the proliferation and migration of HSCs. A and B, cell proliferation analysis of primary HSCs (A) and primary hepatocyte (B) determined by MTT assay. Cells were treated with 40 nM TFF2 for 5 days. Cells in the control group were treated with 0.1% bovine serum albumin PBS. Fresh medium was replaced every 48 h. C, cell proliferation analysis of LX-2 cells treated with indicated concentration of TFF2. D, schematic view of the modiﬁed Boyden chamber assay. E and F, crystal violet staining and quantiﬁcation of primary HSCs migrated to the lower membrane after 4 h treatment. The scale bar represents 500 μm. G, quantiﬁcation of migrated LX-2 cells treated with indicated concentration of TFF2. Cells were isolated from 4-week-old mice, n = 3 to 4. Data are shown as the mean ± SEM. *p < 0.05; **p < 0.01 by unpaired Student’s t test and one-way ANOVA followed by Tukey-adjusted multiple comparisons. HSCs, hepatic stellate cells; TFF2, trefoil factor 2.

Figure Legend Snippet: Figure 5. TFF2 promotes the proliferation and migration of HSCs. A and B, cell proliferation analysis of primary HSCs (A) and primary hepatocyte (B) determined by MTT assay. Cells were treated with 40 nM TFF2 for 5 days. Cells in the control group were treated with 0.1% bovine serum albumin PBS. Fresh medium was replaced every 48 h. C, cell proliferation analysis of LX-2 cells treated with indicated concentration of TFF2. D, schematic view of the modiﬁed Boyden chamber assay. E and F, crystal violet staining and quantiﬁcation of primary HSCs migrated to the lower membrane after 4 h treatment. The scale bar represents 500 μm. G, quantiﬁcation of migrated LX-2 cells treated with indicated concentration of TFF2. Cells were isolated from 4-week-old mice, n = 3 to 4. Data are shown as the mean ± SEM. *p < 0.05; **p < 0.01 by unpaired Student’s t test and one-way ANOVA followed by Tukey-adjusted multiple comparisons. HSCs, hepatic stellate cells; TFF2, trefoil factor 2.

Techniques Used: Migration, MTT Assay, Control, Concentration Assay, Boyden Chamber Assay, Staining, Membrane, Isolation

Figure 6. TFF2 promotes the activation of PDGFRβ signaling. A and B, immunoﬂuorescence imaging of HSCs with recombinant TFF2 treatment. PDGF- bb was used as a positive control. The scale bar represents 25 μm. B, quantiﬁcation of the immunoﬂuorescence images. The ﬂuorescence intensity of p- PDGFRβ/total PDGFRβ was quantiﬁed and then normalized to the control group. n = 30. C, Western blots of primary HSCs treated with 40 nM TFF2 for 5, 15, and 30 min. D, Western blots of primary HSCs treated with different doses of TFF2 for 5 min. Cells were isolated from 4-week-old mice. Data are shown as the mean ± SEM. ***p < 0.001 by one-way ANOVA followed by Tukey-adjusted multiple comparisons. HSCs, hepatic stellate cells; TFF2, trefoil factor 2.

Figure Legend Snippet: Figure 6. TFF2 promotes the activation of PDGFRβ signaling. A and B, immunoﬂuorescence imaging of HSCs with recombinant TFF2 treatment. PDGF- bb was used as a positive control. The scale bar represents 25 μm. B, quantiﬁcation of the immunoﬂuorescence images. The ﬂuorescence intensity of p- PDGFRβ/total PDGFRβ was quantiﬁed and then normalized to the control group. n = 30. C, Western blots of primary HSCs treated with 40 nM TFF2 for 5, 15, and 30 min. D, Western blots of primary HSCs treated with different doses of TFF2 for 5 min. Cells were isolated from 4-week-old mice. Data are shown as the mean ± SEM. ***p < 0.001 by one-way ANOVA followed by Tukey-adjusted multiple comparisons. HSCs, hepatic stellate cells; TFF2, trefoil factor 2.

Techniques Used: Activation Assay, Imaging, Recombinant, Positive Control, Control, Western Blot, Isolation

Figure 7. Increased expression of TFF2 in mice with CCl4-induced liver injury. A, Kaplan–Meier plot for patients with hepatocellular carcinoma. Data source: the cancer genome atlas. Visualization: https://xenabrowser.net/. B, mRNA expression of ﬁbrogenic genes in mice with 1-week vehicle or CCl4 injection. C, Western blotting of liver lysates from mice treated with vehicle or CCl4 for 1 week. D, quantiﬁcations of Western blot images. E, immuno- histochemistry stains of TFF2 in mice treated with vehicle or CCl4 for 3 weeks. The scale bar represents 100 μm. F, working model of the intercellular signaling between OGT-deﬁcient hepatocytes and HSCs. Data are shown as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 by unpaired Student’s t test. HSCs, hepatic stellate cells; OGT, O-GlcNAc transferase; TFF2, trefoil factor 2.

Figure Legend Snippet: Figure 7. Increased expression of TFF2 in mice with CCl4-induced liver injury. A, Kaplan–Meier plot for patients with hepatocellular carcinoma. Data source: the cancer genome atlas. Visualization: https://xenabrowser.net/. B, mRNA expression of ﬁbrogenic genes in mice with 1-week vehicle or CCl4 injection. C, Western blotting of liver lysates from mice treated with vehicle or CCl4 for 1 week. D, quantiﬁcations of Western blot images. E, immuno- histochemistry stains of TFF2 in mice treated with vehicle or CCl4 for 3 weeks. The scale bar represents 100 μm. F, working model of the intercellular signaling between OGT-deﬁcient hepatocytes and HSCs. Data are shown as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 by unpaired Student’s t test. HSCs, hepatic stellate cells; OGT, O-GlcNAc transferase; TFF2, trefoil factor 2.

Techniques Used: Expressing, Injection, Western Blot, Immunohistochemistry

Image Search Results

Journal: Journal of Biological Chemistry

Article Title: Trefoil factor 2 secreted from damaged hepatocytes activates hepatic stellate cells to induce fibrogenesis

doi: 10.1016/j.jbc.2021.100887

Figure Lengend Snippet: Figure 4. The expression and secretion of trefoil factor 2 (TFF2) is increased in OGT-deﬁcient hepatocytes. A, the expression of genes that encode secreted proteins in WT and OGT-LKO livers. Top 50 genes are shown based on the ranks of fold change. B–D, mRNA expression of Tff2 (B), Bmp8b (C), and Gpnmb (D) in primary hepatocytes (PH) and liver lysates. Cell and liver lysates were isolated from 4-week-old WT and OGT-LKO mice. n = 3 to 5. E, Western blotting of proteins extracted from WT and KO hepatocyte-conditioned medium. F, immunoﬂuorescence imaging of primary hepatocytes with the antibody against TFF2 (green) and 40,6-diamidino-2-phenylindole (blue). The scale bar represents 25 μm. G and H, mRNA expression of Tff1 and Tff3 in primary hepatocytes and liver lysates. Cell and liver lysates were isolated from 4-week-old WT and OGT-LKO mice. n = 4 to 5. Data are shown as the mean ± SEM. *p < 0.05; **p < 0.01 by unpaired Student’s t test. HSCs, hepatic stellate cells; OGT, O-GlcNAc transferase; OGT-LKO, liver-speciﬁc OGT KO.

Article Snippet: Recombinant TFF2 (Novus) was dissolved in 0.1% BSA PBS, and the final concentration used was 40 nM.

Techniques: Expressing, Isolation, Western Blot, Imaging

Journal: Journal of Biological Chemistry

Article Title: Trefoil factor 2 secreted from damaged hepatocytes activates hepatic stellate cells to induce fibrogenesis

doi: 10.1016/j.jbc.2021.100887

Figure Lengend Snippet: Figure 5. TFF2 promotes the proliferation and migration of HSCs. A and B, cell proliferation analysis of primary HSCs (A) and primary hepatocyte (B) determined by MTT assay. Cells were treated with 40 nM TFF2 for 5 days. Cells in the control group were treated with 0.1% bovine serum albumin PBS. Fresh medium was replaced every 48 h. C, cell proliferation analysis of LX-2 cells treated with indicated concentration of TFF2. D, schematic view of the modiﬁed Boyden chamber assay. E and F, crystal violet staining and quantiﬁcation of primary HSCs migrated to the lower membrane after 4 h treatment. The scale bar represents 500 μm. G, quantiﬁcation of migrated LX-2 cells treated with indicated concentration of TFF2. Cells were isolated from 4-week-old mice, n = 3 to 4. Data are shown as the mean ± SEM. *p < 0.05; **p < 0.01 by unpaired Student’s t test and one-way ANOVA followed by Tukey-adjusted multiple comparisons. HSCs, hepatic stellate cells; TFF2, trefoil factor 2.

Article Snippet: Recombinant TFF2 (Novus) was dissolved in 0.1% BSA PBS, and the final concentration used was 40 nM.

Techniques: Migration, MTT Assay, Control, Concentration Assay, Boyden Chamber Assay, Staining, Membrane, Isolation

Journal: Journal of Biological Chemistry

Article Title: Trefoil factor 2 secreted from damaged hepatocytes activates hepatic stellate cells to induce fibrogenesis

doi: 10.1016/j.jbc.2021.100887

Figure Lengend Snippet: Figure 6. TFF2 promotes the activation of PDGFRβ signaling. A and B, immunoﬂuorescence imaging of HSCs with recombinant TFF2 treatment. PDGF- bb was used as a positive control. The scale bar represents 25 μm. B, quantiﬁcation of the immunoﬂuorescence images. The ﬂuorescence intensity of p- PDGFRβ/total PDGFRβ was quantiﬁed and then normalized to the control group. n = 30. C, Western blots of primary HSCs treated with 40 nM TFF2 for 5, 15, and 30 min. D, Western blots of primary HSCs treated with different doses of TFF2 for 5 min. Cells were isolated from 4-week-old mice. Data are shown as the mean ± SEM. ***p < 0.001 by one-way ANOVA followed by Tukey-adjusted multiple comparisons. HSCs, hepatic stellate cells; TFF2, trefoil factor 2.

Article Snippet: Recombinant TFF2 (Novus) was dissolved in 0.1% BSA PBS, and the final concentration used was 40 nM.

Techniques: Activation Assay, Imaging, Recombinant, Positive Control, Control, Western Blot, Isolation

Journal: Journal of Biological Chemistry

Article Title: Trefoil factor 2 secreted from damaged hepatocytes activates hepatic stellate cells to induce fibrogenesis

doi: 10.1016/j.jbc.2021.100887

Figure Lengend Snippet: Figure 7. Increased expression of TFF2 in mice with CCl4-induced liver injury. A, Kaplan–Meier plot for patients with hepatocellular carcinoma. Data source: the cancer genome atlas. Visualization: https://xenabrowser.net/. B, mRNA expression of ﬁbrogenic genes in mice with 1-week vehicle or CCl4 injection. C, Western blotting of liver lysates from mice treated with vehicle or CCl4 for 1 week. D, quantiﬁcations of Western blot images. E, immuno- histochemistry stains of TFF2 in mice treated with vehicle or CCl4 for 3 weeks. The scale bar represents 100 μm. F, working model of the intercellular signaling between OGT-deﬁcient hepatocytes and HSCs. Data are shown as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 by unpaired Student’s t test. HSCs, hepatic stellate cells; OGT, O-GlcNAc transferase; TFF2, trefoil factor 2.

Article Snippet: Recombinant TFF2 (Novus) was dissolved in 0.1% BSA PBS, and the final concentration used was 40 nM.

Techniques: Expressing, Injection, Western Blot, Immunohistochemistry

TFF2-Fc treatment requires LINGO3 KO to accelerate healing post DSS-colitis. (A) Change in body weight and (B) disease activity score (DAI) following therapeutic administration of Fc or TFF2-Fc at the timepoints indicated following oral administration of 2.5% DSS in LINGO3 KO and WT mice, as well as (C) Colon length (cm) (D) Inflammation score (E) Mucosal/crypt loss score in tissue collection on day 10. H&E (top) and pERK1/2 (bottom) stained images of colon following (F) control Fc or (G) TFF-Fc treatment 10 days post 2.5% DSS (Magnification 20x) and (H) quantification of pERK+ cells per high powerfield (HPF) for those treatment groups. Red arrowheads indicate pERK1/2+ cells, brown. Mean ± SEM from n=4–6 mice/genotype. 3 independent experiments. * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.0005, **** p ≤ 0.0001

Journal: Scandinavian journal of gastroenterology

Article Title: LINGO3 regulates mucosal tissue regeneration and promotes TFF2 dependent recovery from colitis

doi: 10.1080/00365521.2021.1917650

Figure Lengend Snippet: TFF2-Fc treatment requires LINGO3 KO to accelerate healing post DSS-colitis. (A) Change in body weight and (B) disease activity score (DAI) following therapeutic administration of Fc or TFF2-Fc at the timepoints indicated following oral administration of 2.5% DSS in LINGO3 KO and WT mice, as well as (C) Colon length (cm) (D) Inflammation score (E) Mucosal/crypt loss score in tissue collection on day 10. H&E (top) and pERK1/2 (bottom) stained images of colon following (F) control Fc or (G) TFF-Fc treatment 10 days post 2.5% DSS (Magnification 20x) and (H) quantification of pERK+ cells per high powerfield (HPF) for those treatment groups. Red arrowheads indicate pERK1/2+ cells, brown. Mean ± SEM from n=4–6 mice/genotype. 3 independent experiments. * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.0005, **** p ≤ 0.0001

Article Snippet: Purified murine IgG (RayBiotech), murine recombinant TFF2 (Peprotech) and SDF1a (R&D systems) were labeled with Alexa Fluor 647 dye using the Alexa Fluor 647 protein labeling kit (A30009, Molecular Probes) per manufacturer’s directions.

Techniques: Activity Assay, Staining, Control

TFF2 has weak affinity interactions with LINGO3. (A) Approach for incubation of soluble ligand with transfected HEK cells followed by flow cytometry to identify doubly fluorescent cells. Percent of doubly fluorescent cells identified in the upper right quadrant for LINGO3-GFP transfectants incubated with (B) PBS (C) IgG-Alexa Fluor 647 or (E) TFF2- Alexa Fluor 647. (D) LINGO2-GFP transfected HEK cells incubated with TFF2-Alexa Fluor 647. (F) CXCR4-GFP transfectants incubated with (F) TFF2-Alexa Fluor 647 or (G) SDF1- Alexa Fluor 647, a known high affinity binding partner. (H) Coimmunoprecipitation of Flag-LINGO3 and TFF2-Fc in transfected CHO cells. (I) Control TFF2-Fc and Fc without co-immunoprecipitation migrate at 50 kD and 40 kD.

Journal: Scandinavian journal of gastroenterology

Article Title: LINGO3 regulates mucosal tissue regeneration and promotes TFF2 dependent recovery from colitis

doi: 10.1080/00365521.2021.1917650

Figure Lengend Snippet: TFF2 has weak affinity interactions with LINGO3. (A) Approach for incubation of soluble ligand with transfected HEK cells followed by flow cytometry to identify doubly fluorescent cells. Percent of doubly fluorescent cells identified in the upper right quadrant for LINGO3-GFP transfectants incubated with (B) PBS (C) IgG-Alexa Fluor 647 or (E) TFF2- Alexa Fluor 647. (D) LINGO2-GFP transfected HEK cells incubated with TFF2-Alexa Fluor 647. (F) CXCR4-GFP transfectants incubated with (F) TFF2-Alexa Fluor 647 or (G) SDF1- Alexa Fluor 647, a known high affinity binding partner. (H) Coimmunoprecipitation of Flag-LINGO3 and TFF2-Fc in transfected CHO cells. (I) Control TFF2-Fc and Fc without co-immunoprecipitation migrate at 50 kD and 40 kD.

Techniques: Incubation, Transfection, Flow Cytometry, Binding Assay, Control, Immunoprecipitation

TFF2-Fc mediated signaling cascades are only partially dependent upon LINGO3. Colon tissue from WT or LINGO3 KO mice was collected at 10 days post 2.5% DSS administration combined with TFF2-Fc or Fc treatment and immunostained for (A,B) phosphorylated (p)-STAT3, (C,D) β-catenin, (E,F) proliferative cell marker Ki-67, and (G,H) apoptotic cell marker cleaved caspase 3. (A,C,E,G) are respective representative images (Magnification 20x). (B,F,H) are quantification of + cells per high powerfield for the respective markers and (D) is percent of threshold area for β-catenin. Red arrowheads indicate positively stained cells or tissue regions, in brown. Mean ± SEM from n=4–6 mice/genotype/ 4 images per mouse. 2–3 independent experiments. * p ≤ 0.05, ** p ≤0.005, *** p ≤ 0.0005, **** p ≤ 0.0001

Journal: Scandinavian journal of gastroenterology

Article Title: LINGO3 regulates mucosal tissue regeneration and promotes TFF2 dependent recovery from colitis

doi: 10.1080/00365521.2021.1917650

Figure Lengend Snippet: TFF2-Fc mediated signaling cascades are only partially dependent upon LINGO3. Colon tissue from WT or LINGO3 KO mice was collected at 10 days post 2.5% DSS administration combined with TFF2-Fc or Fc treatment and immunostained for (A,B) phosphorylated (p)-STAT3, (C,D) β-catenin, (E,F) proliferative cell marker Ki-67, and (G,H) apoptotic cell marker cleaved caspase 3. (A,C,E,G) are respective representative images (Magnification 20x). (B,F,H) are quantification of + cells per high powerfield for the respective markers and (D) is percent of threshold area for β-catenin. Red arrowheads indicate positively stained cells or tissue regions, in brown. Mean ± SEM from n=4–6 mice/genotype/ 4 images per mouse. 2–3 independent experiments. * p ≤ 0.05, ** p ≤0.005, *** p ≤ 0.0005, **** p ≤ 0.0001

Techniques: Marker, Staining

LINGO3 deficiency causes perturbations in intestinal stem cell activity. (A) Lgr5 mRNA expression in colons from naïve or day 10 post 2.5% DSS administration. (B) Intestinal organoid culture or (G) colonic organoid culture from WT LINGO3 KO, TFF2 KO mouse strains, brightfield images taken at days 3, 5, 7 (magnification 20x). Arrows point to buds. Number of budding organoids per genotype at day 7 of culture for (C) intestinal organoids and (H) colonic organoids. Intestinal organoid (D) Area (pixels/um) (E) Lgr5, (F) Tff3 and Tff2 mRNA at day 7 of culture. (I) Number of 2D colonic organoids for each genotype. (Mean ± SEM from n=4–6 mice/genotype. 2–3 independent experiments. * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.0005,

Journal: Scandinavian journal of gastroenterology

Article Title: LINGO3 regulates mucosal tissue regeneration and promotes TFF2 dependent recovery from colitis

doi: 10.1080/00365521.2021.1917650

Figure Lengend Snippet: LINGO3 deficiency causes perturbations in intestinal stem cell activity. (A) Lgr5 mRNA expression in colons from naïve or day 10 post 2.5% DSS administration. (B) Intestinal organoid culture or (G) colonic organoid culture from WT LINGO3 KO, TFF2 KO mouse strains, brightfield images taken at days 3, 5, 7 (magnification 20x). Arrows point to buds. Number of budding organoids per genotype at day 7 of culture for (C) intestinal organoids and (H) colonic organoids. Intestinal organoid (D) Area (pixels/um) (E) Lgr5, (F) Tff3 and Tff2 mRNA at day 7 of culture. (I) Number of 2D colonic organoids for each genotype. (Mean ± SEM from n=4–6 mice/genotype. 2–3 independent experiments. * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.0005,

Techniques: Activity Assay, Expressing

Journal: Scandinavian journal of gastroenterology

Article Title: LINGO3 regulates mucosal tissue regeneration and promotes TFF2 dependent recovery from colitis

doi: 10.1080/00365521.2021.1917650

Article Snippet: On day 21, monolayers were wounded in a cross pattern with a sterile pipet tip (1mm width) and treated daily on the apical side with 10 ng/ml recombinant human TFF2 (Peprotech) or 1xPBS (Mock).

Techniques: Activity Assay, Staining, Control

Journal: Scandinavian journal of gastroenterology

Article Title: LINGO3 regulates mucosal tissue regeneration and promotes TFF2 dependent recovery from colitis

doi: 10.1080/00365521.2021.1917650

Techniques: Incubation, Transfection, Flow Cytometry, Binding Assay, Control, Immunoprecipitation

Journal: Scandinavian journal of gastroenterology

Article Title: LINGO3 regulates mucosal tissue regeneration and promotes TFF2 dependent recovery from colitis

doi: 10.1080/00365521.2021.1917650

Techniques: Marker, Staining

Journal: Scandinavian journal of gastroenterology

Article Title: LINGO3 regulates mucosal tissue regeneration and promotes TFF2 dependent recovery from colitis

doi: 10.1080/00365521.2021.1917650

Techniques: Activity Assay, Expressing

Postoperative sleep-restriction (SR) increased myeloid-derived suppressor cells (MDSCs) expansion and decreased splenic CD8 + cells activity via inhibiting splenic trefoil factor 2 (TFF2). ( A ) The expression of TFF2 in the spleen of SR mice with or without dexmedetomidine (Dex) treatment was analysed by real-time PCR (RT-PCR and western blotting. ( B ) Spleen weight in each group after 7 days of SR. ( C ) Flow cytometry analysis of spleen for CD11b + Gr-1 + MDSCs in SR mice with or without Dex treatment. ( D ) The enzyme-linked immunospot (ELISPOT) assay of the levels of interferon-γ (IFN-γ) and Granzyme B (GrB) in splenic CD8 + T cells from SR mice with or without Dex treatment. ( E ) The mRNA and protein expression of TFF2 in the spleen were analysed by RT-PCR and western blotting in SR mice with or without vagus nerve stimulation (VNS). ( F ) Spleen weight in SR mice with the treatment of recombinant human TFF2 protein (rTFF2) or PBS. ( G ) Flow cytometry analysis of spleen for CD11b + Gr-1 + MDSCs in SR mice with the treatment of rTFF2 or PBS. ( H ) ELISPOT assay of the levels of IFN-γ and GrB in splenic CD8 + T cells from SR mice with the treatment of rTFF2 or PBS. All data represent mean ± SEM, n = 5; # P < 0.05, ## P < 0.01.

Journal: Aging (Albany NY)

Article Title: Dexmedetomidine alleviates sleep-restriction-mediated exaggeration of postoperative immunosuppression via splenic TFF2 in aged mice

doi: 10.18632/aging.102952

Figure Lengend Snippet: Postoperative sleep-restriction (SR) increased myeloid-derived suppressor cells (MDSCs) expansion and decreased splenic CD8 + cells activity via inhibiting splenic trefoil factor 2 (TFF2). ( A ) The expression of TFF2 in the spleen of SR mice with or without dexmedetomidine (Dex) treatment was analysed by real-time PCR (RT-PCR and western blotting. ( B ) Spleen weight in each group after 7 days of SR. ( C ) Flow cytometry analysis of spleen for CD11b + Gr-1 + MDSCs in SR mice with or without Dex treatment. ( D ) The enzyme-linked immunospot (ELISPOT) assay of the levels of interferon-γ (IFN-γ) and Granzyme B (GrB) in splenic CD8 + T cells from SR mice with or without Dex treatment. ( E ) The mRNA and protein expression of TFF2 in the spleen were analysed by RT-PCR and western blotting in SR mice with or without vagus nerve stimulation (VNS). ( F ) Spleen weight in SR mice with the treatment of recombinant human TFF2 protein (rTFF2) or PBS. ( G ) Flow cytometry analysis of spleen for CD11b + Gr-1 + MDSCs in SR mice with the treatment of rTFF2 or PBS. ( H ) ELISPOT assay of the levels of IFN-γ and GrB in splenic CD8 + T cells from SR mice with the treatment of rTFF2 or PBS. All data represent mean ± SEM, n = 5; # P < 0.05, ## P < 0.01.

Article Snippet: After 7 days of SR, recombinant human TFF2 protein (rTFF2; 0.2 mg/kg; R&D Systems, Minneapolis, MN, USA) was injected intravenously via the tail vein.

Techniques: Derivative Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry, Enzyme-linked Immunospot, Recombinant

Subdiaphragmatic vagus nerve mediated the attenuated effects of dexmedetomidine (Dex) on sleep-restriction (SR)-induced decrease in splenic trefoil factor 2 (TFF2) expression, increase in the expansion of myeloid-derived suppressor cells (MDSCs) and decrease in CD8 + T cells activity. ( A ) The expression of TFF2 in the spleen of dexmedetomidine (Dex)-treated SR mice with or without bilateral sub-diaphragmatic vagotomy (SDV) was analysed by western blotting. ( B ) spleen weight in Dex-treated SR mice with or without SDV. ( C ) Flow cytometry analysis of spleen for CD11b + Gr-1 + MDSCs in Dex-treated SR mice with or without SDV. ( D ) The enzyme-linked immunospot (ELISPOT) assay of the levels of interferon-γ (IFN-γ) and Granzyme B (GrB) in splenic CD8 + T cells from Dex-treated SR mice with or without SDV. All data represent mean ± SEM, n = 5; # P < 0.05, N.S., not significant.

Journal: Aging (Albany NY)

Article Title: Dexmedetomidine alleviates sleep-restriction-mediated exaggeration of postoperative immunosuppression via splenic TFF2 in aged mice

doi: 10.18632/aging.102952

Figure Lengend Snippet: Subdiaphragmatic vagus nerve mediated the attenuated effects of dexmedetomidine (Dex) on sleep-restriction (SR)-induced decrease in splenic trefoil factor 2 (TFF2) expression, increase in the expansion of myeloid-derived suppressor cells (MDSCs) and decrease in CD8 + T cells activity. ( A ) The expression of TFF2 in the spleen of dexmedetomidine (Dex)-treated SR mice with or without bilateral sub-diaphragmatic vagotomy (SDV) was analysed by western blotting. ( B ) spleen weight in Dex-treated SR mice with or without SDV. ( C ) Flow cytometry analysis of spleen for CD11b + Gr-1 + MDSCs in Dex-treated SR mice with or without SDV. ( D ) The enzyme-linked immunospot (ELISPOT) assay of the levels of interferon-γ (IFN-γ) and Granzyme B (GrB) in splenic CD8 + T cells from Dex-treated SR mice with or without SDV. All data represent mean ± SEM, n = 5; # P < 0.05, N.S., not significant.

Article Snippet: After 7 days of SR, recombinant human TFF2 protein (rTFF2; 0.2 mg/kg; R&D Systems, Minneapolis, MN, USA) was injected intravenously via the tail vein.

Techniques: Expressing, Derivative Assay, Activity Assay, Western Blot, Flow Cytometry, Enzyme-linked Immunospot

Dexmedetomidine (Dex) attenuated postoperative sleep-restriction (SR)-induced decrease in splenic trefoil factor 2 (TFF2) expression, increase in myeloid-derived suppressor cells (MDSCs) expansion and decrease in splenic CD8 + cells activity via improving gut microbiota disturbance. ( A ) Western blotting analysis of splenic TFF2 expression in sham-operated mice and pseudo-germ-free mouse received fecal microbiota transplantation (FMT). ( B ) Spleen weight in sham-operated mice and pseudo-germ-free mouse received FMT. ( C , D ) Flow cytometry analysis of spleen for CD11b + Gr-1 + MDSCs in sham-operated mice and pseudo-germ-free mouse received FMT. ( E ) The enzyme-linked immunospot (ELISPOT) assay of the levels of interferon-γ (IFN-γ) and Granzyme B (GrB) in splenic CD8 + T cells from sham-operated mice and pseudo-germ-free mouse received FMT. All data represent mean ± SEM, n = 5; # P < 0.05, ## P < 0.01.

Journal: Aging (Albany NY)

Article Title: Dexmedetomidine alleviates sleep-restriction-mediated exaggeration of postoperative immunosuppression via splenic TFF2 in aged mice

doi: 10.18632/aging.102952

Figure Lengend Snippet: Dexmedetomidine (Dex) attenuated postoperative sleep-restriction (SR)-induced decrease in splenic trefoil factor 2 (TFF2) expression, increase in myeloid-derived suppressor cells (MDSCs) expansion and decrease in splenic CD8 + cells activity via improving gut microbiota disturbance. ( A ) Western blotting analysis of splenic TFF2 expression in sham-operated mice and pseudo-germ-free mouse received fecal microbiota transplantation (FMT). ( B ) Spleen weight in sham-operated mice and pseudo-germ-free mouse received FMT. ( C , D ) Flow cytometry analysis of spleen for CD11b + Gr-1 + MDSCs in sham-operated mice and pseudo-germ-free mouse received FMT. ( E ) The enzyme-linked immunospot (ELISPOT) assay of the levels of interferon-γ (IFN-γ) and Granzyme B (GrB) in splenic CD8 + T cells from sham-operated mice and pseudo-germ-free mouse received FMT. All data represent mean ± SEM, n = 5; # P < 0.05, ## P < 0.01.

Article Snippet: After 7 days of SR, recombinant human TFF2 protein (rTFF2; 0.2 mg/kg; R&D Systems, Minneapolis, MN, USA) was injected intravenously via the tail vein.

Techniques: Expressing, Derivative Assay, Activity Assay, Western Blot, Transplantation Assay, Flow Cytometry, Enzyme-linked Immunospot

Subdiaphragmatic vagus nerve served as a bridge between gut microbiota and spleen after postoperative sleep-restriction (SR). ( A ) Western blotting analysis of splenic trefoil factor 2 (TFF2) expression in pseudo-germ-free mouse received fecal microbiota transplantation (FMT) with feces of SR mice, dexmedetomidine (Dex)-treated SR mice or Dex-treated SR mice with sub-diaphragmatic vagotomy (SDV). ( B ) Spleen weight in pseudo-germ-free mouse received FMT with feces of SR mice, Dex-treated SR mice or Dex-treated SR mice with SDV. ( C ) Flow cytometry analysis of spleen for CD11b + Gr-1 + myeloid-derived suppressor cells (MDSCs) in pseudo-germ-free mouse received FMT with feces of SR mice, Dex-treated SR mice or Dex-treated SR mice with SDV. ( D ) The enzyme-linked immunospot (ELISPOT) assay of the levels of interferon-γ (IFN-γ) and Granzyme B (GrB) in splenic CD8 + T cells from pseudo-germ-free mouse received FMT with feces of SR mice, Dex-treated SR mice or Dex-treated SR mice with SDV. All data represent mean ± SEM, n = 5; # P < 0.05, ## P < 0.01.

Journal: Aging (Albany NY)

Article Title: Dexmedetomidine alleviates sleep-restriction-mediated exaggeration of postoperative immunosuppression via splenic TFF2 in aged mice

doi: 10.18632/aging.102952

Figure Lengend Snippet: Subdiaphragmatic vagus nerve served as a bridge between gut microbiota and spleen after postoperative sleep-restriction (SR). ( A ) Western blotting analysis of splenic trefoil factor 2 (TFF2) expression in pseudo-germ-free mouse received fecal microbiota transplantation (FMT) with feces of SR mice, dexmedetomidine (Dex)-treated SR mice or Dex-treated SR mice with sub-diaphragmatic vagotomy (SDV). ( B ) Spleen weight in pseudo-germ-free mouse received FMT with feces of SR mice, Dex-treated SR mice or Dex-treated SR mice with SDV. ( C ) Flow cytometry analysis of spleen for CD11b + Gr-1 + myeloid-derived suppressor cells (MDSCs) in pseudo-germ-free mouse received FMT with feces of SR mice, Dex-treated SR mice or Dex-treated SR mice with SDV. ( D ) The enzyme-linked immunospot (ELISPOT) assay of the levels of interferon-γ (IFN-γ) and Granzyme B (GrB) in splenic CD8 + T cells from pseudo-germ-free mouse received FMT with feces of SR mice, Dex-treated SR mice or Dex-treated SR mice with SDV. All data represent mean ± SEM, n = 5; # P < 0.05, ## P < 0.01.

Article Snippet: After 7 days of SR, recombinant human TFF2 protein (rTFF2; 0.2 mg/kg; R&D Systems, Minneapolis, MN, USA) was injected intravenously via the tail vein.

Techniques: Western Blot, Expressing, Transplantation Assay, Flow Cytometry, Derivative Assay, Enzyme-linked Immunospot

Splenic trefoil factor 2 (TFF2) was essential to attenuate SR-induced reduced protective ability against Escherichia coli ( E. coli ) pneumonia, increased expression of IL-4 and IL-13 in the lung and M2 polarization of alveolar macrophages (AMs), and decreased phagocytic activity of AMs. ( A ) Enumeration of colony-forming units (CFU) per milliliter of bronchoalveolar lavage analyzed one day after E. coli pneumonia in postoperative SR mice treated with recombinant human TFF2 protein (rTFF2) or PBS. ( B ) ELISA determination of the concentrations of IL-4 and IL-13 in the lungs of postoperative SR mice treated with rTFF2 or PBS one day after E. coli pneumonia. ( C ) The phagocytic activity of alveolar macrophages from postoperative SR mice treated with rTFF2 or PBS. ( D ) Real-time PCR (RT-PCR) analysis of the mRNA expression of Arg1, YM and iNOS in alveolar macrophages from postoperative SR mice treated with rTFF2 or PBS. All data represent mean ± SEM, n = 5; # P < 0.05, ## P < 0.01.

Journal: Aging (Albany NY)

Article Title: Dexmedetomidine alleviates sleep-restriction-mediated exaggeration of postoperative immunosuppression via splenic TFF2 in aged mice

doi: 10.18632/aging.102952

Figure Lengend Snippet: Splenic trefoil factor 2 (TFF2) was essential to attenuate SR-induced reduced protective ability against Escherichia coli ( E. coli ) pneumonia, increased expression of IL-4 and IL-13 in the lung and M2 polarization of alveolar macrophages (AMs), and decreased phagocytic activity of AMs. ( A ) Enumeration of colony-forming units (CFU) per milliliter of bronchoalveolar lavage analyzed one day after E. coli pneumonia in postoperative SR mice treated with recombinant human TFF2 protein (rTFF2) or PBS. ( B ) ELISA determination of the concentrations of IL-4 and IL-13 in the lungs of postoperative SR mice treated with rTFF2 or PBS one day after E. coli pneumonia. ( C ) The phagocytic activity of alveolar macrophages from postoperative SR mice treated with rTFF2 or PBS. ( D ) Real-time PCR (RT-PCR) analysis of the mRNA expression of Arg1, YM and iNOS in alveolar macrophages from postoperative SR mice treated with rTFF2 or PBS. All data represent mean ± SEM, n = 5; # P < 0.05, ## P < 0.01.

Article Snippet: After 7 days of SR, recombinant human TFF2 protein (rTFF2; 0.2 mg/kg; R&D Systems, Minneapolis, MN, USA) was injected intravenously via the tail vein.

Techniques: Expressing, Activity Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Schematics illustrating the signaling mechanisms of sleep-restriction in postoperative immunosuppression and its treatment by dexmedetomidine. Sleep-restriction exaggerates intestinal flora disorder, furtherly decreases splenic trefoil factor 2 (TFF2) expression, which subsequently leads to the increase in myeloid-derived suppressor cells (MDSCs) numbers and decrease in splenic CD8 + T cells activity. Subdiaphragmatic vagus nerve (SVN) served as an important conduit of gut microbiota-spleen communication. SR-induced exaggeration of postoperative immunosuppression was characterized by increased expression of IL-4 and IL-13 in the lung, increased M2 polarization of alveolar macrophages (AMs), decreased phagocytic activity of AMs and thus decreased antimicrobial activity in E. coli pneumonia. Dexmedetomidine treatment during SR alleviated SR-induced decrease in postoperative immunosuppression through gut microbiota and SVN.

Journal: Aging (Albany NY)

Article Title: Dexmedetomidine alleviates sleep-restriction-mediated exaggeration of postoperative immunosuppression via splenic TFF2 in aged mice

doi: 10.18632/aging.102952

Figure Lengend Snippet: Schematics illustrating the signaling mechanisms of sleep-restriction in postoperative immunosuppression and its treatment by dexmedetomidine. Sleep-restriction exaggerates intestinal flora disorder, furtherly decreases splenic trefoil factor 2 (TFF2) expression, which subsequently leads to the increase in myeloid-derived suppressor cells (MDSCs) numbers and decrease in splenic CD8 + T cells activity. Subdiaphragmatic vagus nerve (SVN) served as an important conduit of gut microbiota-spleen communication. SR-induced exaggeration of postoperative immunosuppression was characterized by increased expression of IL-4 and IL-13 in the lung, increased M2 polarization of alveolar macrophages (AMs), decreased phagocytic activity of AMs and thus decreased antimicrobial activity in E. coli pneumonia. Dexmedetomidine treatment during SR alleviated SR-induced decrease in postoperative immunosuppression through gut microbiota and SVN.

Article Snippet: After 7 days of SR, recombinant human TFF2 protein (rTFF2; 0.2 mg/kg; R&D Systems, Minneapolis, MN, USA) was injected intravenously via the tail vein.

Techniques: Expressing, Derivative Assay, Activity Assay

The sequences of primers for real-time PCR.

Journal: Aging (Albany NY)

Article Title: Dexmedetomidine alleviates sleep-restriction-mediated exaggeration of postoperative immunosuppression via splenic TFF2 in aged mice

doi: 10.18632/aging.102952

Figure Lengend Snippet: The sequences of primers for real-time PCR.

Article Snippet: After 7 days of SR, recombinant human TFF2 protein (rTFF2; 0.2 mg/kg; R&D Systems, Minneapolis, MN, USA) was injected intravenously via the tail vein.

Techniques:

$Analysis of human gastric extract (M C -577). ( A ) Elution profile after SEC on a Superdex 75 HL column as determined by absorbance at 280 nm (PAS-positive mucin fractions: pink). Underneath: Distribution of the relative trefoil factor family 2 (TFF2) content as determined by Western blot analysis under reducing conditions and semi-quantitative analysis of the typical 20 k-band intensities. ( B ) 1% agarose gel electrophoresis (AgGE) and subsequent Western blot analysis of the fractions B7-C2 concerning TFF2, MUC6 (lectin GSA-II), and MUC5AC, respectively. The start is marked with a dot on the left. ( C ) Fifteen percent SDS-PAGE and subsequent Western blot analysis of fractions B10/B11 and D2/D3 concerning TFF2 (R, NR: reducing and non-reducing conditions, respectively). The molecular mass standard is indicated left. ( D ) Digestion of fraction B12 with PNGase F and Western blot analysis concerning TFF2 (15% SDS-PAGE, reducing conditions); c, undigested control; F, sample digested with PNGase F.$

Journal: International Journal of Molecular Sciences

Article Title: Different Forms of TFF2, A Lectin of the Human Gastric Mucus Barrier: In Vitro Binding Studies

doi: 10.3390/ijms20235871

Figure Lengend Snippet: Analysis of human gastric extract (M C -577). ( A ) Elution profile after SEC on a Superdex 75 HL column as determined by absorbance at 280 nm (PAS-positive mucin fractions: pink). Underneath: Distribution of the relative trefoil factor family 2 (TFF2) content as determined by Western blot analysis under reducing conditions and semi-quantitative analysis of the typical 20 k-band intensities. ( B ) 1% agarose gel electrophoresis (AgGE) and subsequent Western blot analysis of the fractions B7-C2 concerning TFF2, MUC6 (lectin GSA-II), and MUC5AC, respectively. The start is marked with a dot on the left. ( C ) Fifteen percent SDS-PAGE and subsequent Western blot analysis of fractions B10/B11 and D2/D3 concerning TFF2 (R, NR: reducing and non-reducing conditions, respectively). The molecular mass standard is indicated left. ( D ) Digestion of fraction B12 with PNGase F and Western blot analysis concerning TFF2 (15% SDS-PAGE, reducing conditions); c, undigested control; F, sample digested with PNGase F.

Article Snippet: TFF2 from the porcine pancreas, as well as recombinant human TFF2 (glycosylated and non-glycosylated forms), were kindly provided by Dr. L. Thim (Novo Nordisk A/S, Maaloev, Denmark) [ , ].

Techniques: Western Blot, Agarose Gel Electrophoresis, SDS Page, Control

$Relative TFF2 content in the high- and low-molecular-mass regions after non-reducing SDS-PAGE (Mc-577). ( A,B ) TFF2-containing fraction B12 after SEC of a human gastric extract (from ) was subjected to non-reducing 15% SDS-PAGE: Western blot analysis concerning TFF2 (A); Coomassie-stained part of the gel (B). Then, the high- (H) and low-molecular-mass regions (L1–L3) were cut out as indicated, proteins were eluted, and subjected to reducing SDS-PAGE. In addition, the remaining high-molecular-mass-samples not entering the gel were removed from the gel pockets (P). ( C ) Western blot analysis concerning TFF2 of P, H, and L1–L3 (reducing conditions).$

Journal: International Journal of Molecular Sciences

Article Title: Different Forms of TFF2, A Lectin of the Human Gastric Mucus Barrier: In Vitro Binding Studies

doi: 10.3390/ijms20235871

Figure Lengend Snippet: Relative TFF2 content in the high- and low-molecular-mass regions after non-reducing SDS-PAGE (Mc-577). ( A,B ) TFF2-containing fraction B12 after SEC of a human gastric extract (from ) was subjected to non-reducing 15% SDS-PAGE: Western blot analysis concerning TFF2 (A); Coomassie-stained part of the gel (B). Then, the high- (H) and low-molecular-mass regions (L1–L3) were cut out as indicated, proteins were eluted, and subjected to reducing SDS-PAGE. In addition, the remaining high-molecular-mass-samples not entering the gel were removed from the gel pockets (P). ( C ) Western blot analysis concerning TFF2 of P, H, and L1–L3 (reducing conditions).

Techniques: SDS Page, Western Blot, Staining

$Analysis of human gastric extracts after boiling with SDS/EDTA (M C -406A) or after TRIzol ® extraction (M C -577), respectively. ( A ) High-molecular-mass TFF2 was purified via SEC from the human gastric corpus, similar to as shown in . Then, the corresponding fractions (B6–B10) were boiled with 1% SDS + 30 mM EDTA and separated immediately on a Superdex 75 HL column again. Elution profile after SEC as determined by absorbance at 280 nm (PAS-positive mucin fractions: pink). Underneath: Distribution of the relative TFF2 content. ( B ) Alternatively, a human gastric corpus specimen was extracted with TRIzol ® . Elution profile after SEC on a Superdex 75 HL column (PAS-positive fractions: pink). Underneath: Distribution of the relative TFF2 content. ( C , D ) Western blot analysis concerning TFF2 (15% SDS-PAGE), MUC6 (lectin GSA-II, 1% AgGE), and MUC5AC (1% AgGE), respectively, of the SDS/EDTA extract ( C ) or the TRIzol ® extract ( D ). Shown are also the hybridization signals (autoradiography) obtained after incubating the blots with 125 I-labeled recombinant human non-glycosylated TFF2 ( C ) or porcine pancreatic TFF2 ( D ) (overlay assays). The start of the AgGE is marked with a dot on the left.$

Journal: International Journal of Molecular Sciences

Article Title: Different Forms of TFF2, A Lectin of the Human Gastric Mucus Barrier: In Vitro Binding Studies

doi: 10.3390/ijms20235871

Figure Lengend Snippet: Analysis of human gastric extracts after boiling with SDS/EDTA (M C -406A) or after TRIzol ® extraction (M C -577), respectively. ( A ) High-molecular-mass TFF2 was purified via SEC from the human gastric corpus, similar to as shown in . Then, the corresponding fractions (B6–B10) were boiled with 1% SDS + 30 mM EDTA and separated immediately on a Superdex 75 HL column again. Elution profile after SEC as determined by absorbance at 280 nm (PAS-positive mucin fractions: pink). Underneath: Distribution of the relative TFF2 content. ( B ) Alternatively, a human gastric corpus specimen was extracted with TRIzol ® . Elution profile after SEC on a Superdex 75 HL column (PAS-positive fractions: pink). Underneath: Distribution of the relative TFF2 content. ( C , D ) Western blot analysis concerning TFF2 (15% SDS-PAGE), MUC6 (lectin GSA-II, 1% AgGE), and MUC5AC (1% AgGE), respectively, of the SDS/EDTA extract ( C ) or the TRIzol ® extract ( D ). Shown are also the hybridization signals (autoradiography) obtained after incubating the blots with 125 I-labeled recombinant human non-glycosylated TFF2 ( C ) or porcine pancreatic TFF2 ( D ) (overlay assays). The start of the AgGE is marked with a dot on the left.

Techniques: Extraction, Purification, Western Blot, SDS Page, Hybridization, Autoradiography, Labeling, Recombinant

$Stepwise extraction of a human gastric corpus specimen (M C -577) and analysis of the extracts E0 and E1. ( A,B ) Elution profiles of extract E0 (A) and E1 (B), respectively, after SEC on a Superdex 75 HL column as determined by absorbance at 280 nm (PAS-positive mucin fractions: pink). Underneath: Distribution of the relative TFF2 content. ( C ) 1% AgGE and subsequent Western blot analysis of the high-molecular-mass fractions B5-C3 concerning MUC6 (lectin GSA-II), MUC5AC, and TFF2. ( D ) 15% SDS-PAGE under reducing (R) or non-reducing conditions (NR) of low- and high-molecular-mass fractions and Western blot analysis concerning TFF2 (post-in-gel reduction).$

Journal: International Journal of Molecular Sciences

Article Title: Different Forms of TFF2, A Lectin of the Human Gastric Mucus Barrier: In Vitro Binding Studies

doi: 10.3390/ijms20235871

Figure Lengend Snippet: Stepwise extraction of a human gastric corpus specimen (M C -577) and analysis of the extracts E0 and E1. ( A,B ) Elution profiles of extract E0 (A) and E1 (B), respectively, after SEC on a Superdex 75 HL column as determined by absorbance at 280 nm (PAS-positive mucin fractions: pink). Underneath: Distribution of the relative TFF2 content. ( C ) 1% AgGE and subsequent Western blot analysis of the high-molecular-mass fractions B5-C3 concerning MUC6 (lectin GSA-II), MUC5AC, and TFF2. ( D ) 15% SDS-PAGE under reducing (R) or non-reducing conditions (NR) of low- and high-molecular-mass fractions and Western blot analysis concerning TFF2 (post-in-gel reduction).

Techniques: Extraction, Western Blot, SDS Page

$Analysis of a human gastric corpus E1-extract (M C -577) after reduction in boiling 1% β-mercaptoethanol. ( A ) Elution profiles after SEC on a Sephacryl S-500 High Resolution column as determined by absorbance at 280 nm. Underneath: Distribution of the relative TFF2 content. For comparison, the fractions were analyzed for their mucin content with the help of the PAS reaction (pink). ( B ) 1% AgGE and subsequent Western blot analysis of the mucin containing fractions B8-D12. The start is marked with a dot on the left. Shown are the reactivities for MUC6 (lectin GSA-II), MUC5AC, and the hybridization signals (autoradiography) obtained after incubating the blot with 125 I-labeled porcine pancreatic TFF2 (pTFF2) or 125 I-labeled recombinant human glycosylated TFF2 (TFF2 glyc ; overlay assays).$

Journal: International Journal of Molecular Sciences

Article Title: Different Forms of TFF2, A Lectin of the Human Gastric Mucus Barrier: In Vitro Binding Studies

doi: 10.3390/ijms20235871

Figure Lengend Snippet: Analysis of a human gastric corpus E1-extract (M C -577) after reduction in boiling 1% β-mercaptoethanol. ( A ) Elution profiles after SEC on a Sephacryl S-500 High Resolution column as determined by absorbance at 280 nm. Underneath: Distribution of the relative TFF2 content. For comparison, the fractions were analyzed for their mucin content with the help of the PAS reaction (pink). ( B ) 1% AgGE and subsequent Western blot analysis of the mucin containing fractions B8-D12. The start is marked with a dot on the left. Shown are the reactivities for MUC6 (lectin GSA-II), MUC5AC, and the hybridization signals (autoradiography) obtained after incubating the blot with 125 I-labeled porcine pancreatic TFF2 (pTFF2) or 125 I-labeled recombinant human glycosylated TFF2 (TFF2 glyc ; overlay assays).

Techniques: Comparison, Western Blot, Hybridization, Autoradiography, Labeling, Recombinant

$In vitro binding studies with 125 I-labeled recombinant human glycosylated TFF2 (overlay assays). Human gastric mucin preparations (Mc-577) were depleted from TFF2 by reduction and purified via SEC on Sephacryl S-500 High Resolution . Then, the PAS-positive mucin fractions B12/C1 were separated on 1% AgGE and subsequently blotted onto nitrocellulose. The start is marked with a dot on the left. Shown are the hybridization signals (autoradiography) obtained after incubating the blots with 125 I-labeled TFF2 (overlay assay of fractions B12/C1) in the presence of 2.5 mM Ca 2+ unless indicated otherwise. Underneath: Semi-quantitative analysis of the intensities (the relative amount of 125 I-TFF2 bound in lanes B12/C1). ( A ) Hybridization at various Ca 2+ concentrations: in the presence of 2.5 mM Ca 2+ , 10 mM Ca 2+ , 30 mM EDTA (and no added Ca 2+ ), and no added Ca 2+ . ( B ) Hybridization after competition with the lectin GSA-II, and after competition with the antibody HIK1083. ( C ) Hybridization in the presence of 1 mM methyl-α-D-galactopyranoside (αGal), and in the presence of 1 mM methyl-β-D-galactopyranoside (βGal).$

Journal: International Journal of Molecular Sciences

Article Title: Different Forms of TFF2, A Lectin of the Human Gastric Mucus Barrier: In Vitro Binding Studies

doi: 10.3390/ijms20235871

Figure Lengend Snippet: In vitro binding studies with 125 I-labeled recombinant human glycosylated TFF2 (overlay assays). Human gastric mucin preparations (Mc-577) were depleted from TFF2 by reduction and purified via SEC on Sephacryl S-500 High Resolution . Then, the PAS-positive mucin fractions B12/C1 were separated on 1% AgGE and subsequently blotted onto nitrocellulose. The start is marked with a dot on the left. Shown are the hybridization signals (autoradiography) obtained after incubating the blots with 125 I-labeled TFF2 (overlay assay of fractions B12/C1) in the presence of 2.5 mM Ca 2+ unless indicated otherwise. Underneath: Semi-quantitative analysis of the intensities (the relative amount of 125 I-TFF2 bound in lanes B12/C1). ( A ) Hybridization at various Ca 2+ concentrations: in the presence of 2.5 mM Ca 2+ , 10 mM Ca 2+ , 30 mM EDTA (and no added Ca 2+ ), and no added Ca 2+ . ( B ) Hybridization after competition with the lectin GSA-II, and after competition with the antibody HIK1083. ( C ) Hybridization in the presence of 1 mM methyl-α-D-galactopyranoside (αGal), and in the presence of 1 mM methyl-β-D-galactopyranoside (βGal).

Techniques: In Vitro, Binding Assay, Labeling, Recombinant, Purification, Hybridization, Autoradiography, Overlay Assay

Direct binding of 125 I-labeled recombinant human glycosylated TFF2 and a commercial porcine gastric mucin preparation (S-mucin) in solution. ( A ) 125 I-labeled recombinant human glycosylated TFF2 was directly incubated with the S-mucin preparation, separated by 15% SDS-PAGE under non-reducing (NR) or reducing conditions (R), and the 125 I-labeled recombinant human glycosylated TFF2 was detected by Western blotting and autoradiography. Lanes 1, 2, 4, 6, 7: mixture of 125 I-labeled TFF2 and S-mucin; lanes 3, 5: 125 I-labeled TFF2 without S-mucin. ( B ) The relative amount of 125 I-labeled recombinant human glycosylated TFF2, which remained in the gel pockets after electrophoresis.

Journal: International Journal of Molecular Sciences

Article Title: Different Forms of TFF2, A Lectin of the Human Gastric Mucus Barrier: In Vitro Binding Studies

doi: 10.3390/ijms20235871

Figure Lengend Snippet: Direct binding of 125 I-labeled recombinant human glycosylated TFF2 and a commercial porcine gastric mucin preparation (S-mucin) in solution. ( A ) 125 I-labeled recombinant human glycosylated TFF2 was directly incubated with the S-mucin preparation, separated by 15% SDS-PAGE under non-reducing (NR) or reducing conditions (R), and the 125 I-labeled recombinant human glycosylated TFF2 was detected by Western blotting and autoradiography. Lanes 1, 2, 4, 6, 7: mixture of 125 I-labeled TFF2 and S-mucin; lanes 3, 5: 125 I-labeled TFF2 without S-mucin. ( B ) The relative amount of 125 I-labeled recombinant human glycosylated TFF2, which remained in the gel pockets after electrophoresis.

Techniques: Binding Assay, Labeling, Recombinant, Incubation, SDS Page, Western Blot, Autoradiography, Electrophoresis

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.

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: Microinjection 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

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.

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.

Techniques: Imaging, Staining, Control

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.

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.

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

Elastase-Cre-mediated Pdx1 inactivation reduces acinar TFF2 in embryonic and neonatal pancreas. ( A ) The expression of TFF2 was detected by RT-PCR in control mice pancreas from E16.5. The original data are shown in Supplementary Fig. . ( B ) Expression of TFF2 is significantly less in Pdx1cKO mice (red) than in control mice (blue). (control mice: n = 7 at E14.5, n = 5 at E16.5, n = 5 at E18.5, and n = 7 at P1; Pdx1cKO mice: n = 5 at E14.5, n = 6 at E16.5, n = 6 at E18.5, and n = 7 at P1; p = N.D at E14.5, p = 0.041 at E16.5, p = 0.0065 at E18.5 and p = 0.0040 at P1). Note that the expression of TFF2 in the mutant stomach is equivalent to that in control stomach at P1 (right panel) (control mice, n = 3, Pdx1cKOmice, n = 3, p = 0.68122). ( C ) Immunostaining of TFF2. TFF2 expression was detected in exocrine cells including the proximal (dotted lines) and distal ducts and acinar cells, but not in islets (arrows) in control mice (upper panels). In Pdx1cKO mice, TFF2 expression was hardly detectable except in the proximal ducts (dotted lines), which were not recombined by Elastase-Cre (bottom panels). These expression patterns were confirmed in at least three individual mice for both genotypes. Scale bars, 100 μm. Bars represent the mean value ± SE. *p < 0.05, **p < 0.01.

Journal: Scientific Reports

Article Title: Exocrine tissue-driven TFF2 prevents apoptotic cell death of endocrine lineage during pancreas organogenesis

doi: 10.1038/s41598-018-38062-9

Figure Lengend Snippet: Elastase-Cre-mediated Pdx1 inactivation reduces acinar TFF2 in embryonic and neonatal pancreas. ( A ) The expression of TFF2 was detected by RT-PCR in control mice pancreas from E16.5. The original data are shown in Supplementary Fig. . ( B ) Expression of TFF2 is significantly less in Pdx1cKO mice (red) than in control mice (blue). (control mice: n = 7 at E14.5, n = 5 at E16.5, n = 5 at E18.5, and n = 7 at P1; Pdx1cKO mice: n = 5 at E14.5, n = 6 at E16.5, n = 6 at E18.5, and n = 7 at P1; p = N.D at E14.5, p = 0.041 at E16.5, p = 0.0065 at E18.5 and p = 0.0040 at P1). Note that the expression of TFF2 in the mutant stomach is equivalent to that in control stomach at P1 (right panel) (control mice, n = 3, Pdx1cKOmice, n = 3, p = 0.68122). ( C ) Immunostaining of TFF2. TFF2 expression was detected in exocrine cells including the proximal (dotted lines) and distal ducts and acinar cells, but not in islets (arrows) in control mice (upper panels). In Pdx1cKO mice, TFF2 expression was hardly detectable except in the proximal ducts (dotted lines), which were not recombined by Elastase-Cre (bottom panels). These expression patterns were confirmed in at least three individual mice for both genotypes. Scale bars, 100 μm. Bars represent the mean value ± SE. *p < 0.05, **p < 0.01.

Article Snippet: Recombinant mouse TFF2 (#RPA748MU01) was purchased from Uscn Life Science (Texas, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Mutagenesis, Immunostaining

TFF2 prevents apoptosis of embryonic insulin-expressing cells through CXCR4. Explant culture of E16.5 pancreatic tissue for 48 hours. ( A ) Immunostaining of insulin (red), beta-catenin (green) and DAPI (blue) for the cultured explant without reagents (left panel), with rTFF2 (middle panel) and with rTFF2 plus AMD3100 (right panel). ( B ) Quantitative analysis of the number of Insulin+ cells. ( C ) Immunostaining of pHH3 (green), insulin (red) and DAPI (blue) for cultured control explant (left panel) and for Pdx1cKO explant without and with rTFF2 (middle and right panels, respectively). The yellow arrowhead shows pHH3-positive Insulin+ cells. ( D ) Quantitative analyses of Insulin+ cell proliferation. ( E ) Immunostaining of TUNEL (green), insulin (red) and DAPI (blue) for cultured Pdx1cKO explant without reagents (left panel), with rTFF2 (middle panel) and with rTFF2 plus AMD3100 (right panel). Yellow arrowheads show TUNEL-positive Insulin+ cells. ( F ) Quantitative analysis of Insulin+ cell apoptosis in Pdx1cKO explant. In control mice pancreas, CXCR4 protected Insulin+ cells from apoptosis. ( G ) Immunostaining of TUNEL (green), insulin (red) and DAPI (blue) for the control explant without and with AMD3100 (left and right panels, respectively). Yellow arrowheads show TUNEL-positive Insulin+ cells. ( H ) Quantitative analyses of Insulin+ cell apoptosis. Scale bars, 100 μm. Bars represent the mean value ± SE. *p < 0.05, **p < 0.01.

Journal: Scientific Reports

Article Title: Exocrine tissue-driven TFF2 prevents apoptotic cell death of endocrine lineage during pancreas organogenesis

doi: 10.1038/s41598-018-38062-9

Figure Lengend Snippet: TFF2 prevents apoptosis of embryonic insulin-expressing cells through CXCR4. Explant culture of E16.5 pancreatic tissue for 48 hours. ( A ) Immunostaining of insulin (red), beta-catenin (green) and DAPI (blue) for the cultured explant without reagents (left panel), with rTFF2 (middle panel) and with rTFF2 plus AMD3100 (right panel). ( B ) Quantitative analysis of the number of Insulin+ cells. ( C ) Immunostaining of pHH3 (green), insulin (red) and DAPI (blue) for cultured control explant (left panel) and for Pdx1cKO explant without and with rTFF2 (middle and right panels, respectively). The yellow arrowhead shows pHH3-positive Insulin+ cells. ( D ) Quantitative analyses of Insulin+ cell proliferation. ( E ) Immunostaining of TUNEL (green), insulin (red) and DAPI (blue) for cultured Pdx1cKO explant without reagents (left panel), with rTFF2 (middle panel) and with rTFF2 plus AMD3100 (right panel). Yellow arrowheads show TUNEL-positive Insulin+ cells. ( F ) Quantitative analysis of Insulin+ cell apoptosis in Pdx1cKO explant. In control mice pancreas, CXCR4 protected Insulin+ cells from apoptosis. ( G ) Immunostaining of TUNEL (green), insulin (red) and DAPI (blue) for the control explant without and with AMD3100 (left and right panels, respectively). Yellow arrowheads show TUNEL-positive Insulin+ cells. ( H ) Quantitative analyses of Insulin+ cell apoptosis. Scale bars, 100 μm. Bars represent the mean value ± SE. *p < 0.05, **p < 0.01.

Article Snippet: Recombinant mouse TFF2 (#RPA748MU01) was purchased from Uscn Life Science (Texas, USA).

Techniques: Expressing, Immunostaining, Cell Culture, Control, TUNEL Assay

Nkx6.1-positive trunk cells are affected by TFF2. Analyses of Nkx6.1+/Insulin- trunk cells in explant culture experiments. ( A ) Immunostaining of Nkx6.1 (green) and insulin (red) for cultured Pdx1cKO explant without reagents (left panel), with rTFF2 (middle panel) and with rTFF2 plus AMD3100 (right panel). ( B ) Quantitative analyses of the number of Nkx6.1+/Insulin- trunk cells. ( C ) TUNEL staining of cultured Pdx1cKO explant without and with rTFF2 (left and right panels, respectively). ( D ) Quantitative analyses of Nkx6.1+/Insulin- trunk cell apoptosis. Scale bars, 100 μm. Bars represent the mean value ± SE. *p < 0.05.

Journal: Scientific Reports

Article Title: Exocrine tissue-driven TFF2 prevents apoptotic cell death of endocrine lineage during pancreas organogenesis

doi: 10.1038/s41598-018-38062-9

Figure Lengend Snippet: Nkx6.1-positive trunk cells are affected by TFF2. Analyses of Nkx6.1+/Insulin- trunk cells in explant culture experiments. ( A ) Immunostaining of Nkx6.1 (green) and insulin (red) for cultured Pdx1cKO explant without reagents (left panel), with rTFF2 (middle panel) and with rTFF2 plus AMD3100 (right panel). ( B ) Quantitative analyses of the number of Nkx6.1+/Insulin- trunk cells. ( C ) TUNEL staining of cultured Pdx1cKO explant without and with rTFF2 (left and right panels, respectively). ( D ) Quantitative analyses of Nkx6.1+/Insulin- trunk cell apoptosis. Scale bars, 100 μm. Bars represent the mean value ± SE. *p < 0.05.

Article Snippet: Recombinant mouse TFF2 (#RPA748MU01) was purchased from Uscn Life Science (Texas, USA).

Techniques: Immunostaining, Cell Culture, TUNEL Assay, Staining

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