recombinant human tff2 protein  (R&D Systems)

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

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

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

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

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

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

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

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:

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

Journal: The Journal of Physiology

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

doi: 10.1113/JP277259

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

Article Snippet: 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

Journal: The Journal of Physiology

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

doi: 10.1113/JP277259

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

Article Snippet: 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: Fluorescence, Control, Comparison, Membrane, Injection, Microinjection