Journal: PLoS ONE

Article Title: TL1A Increases Expression of CD25, LFA-1, CD134 and CD154, and Induces IL-22 and GM-CSF Production from Effector CD4 T-Cells

doi: 10.1371/journal.pone.0105627

Figure Lengend Snippet: After 12 days, beads were removed and the cells set up in new media. Cytokines were added in the following concentrations IL-12: 4 ng/mL, IL-15: 10 ng/mL, IL-18: 40 ng/mL, TL1A: 100 ng/mL, TL1A Ab: 1 µg/mL, CsA 1 µg/mL. Pictures using the light microscope were taken at 25× magnification.

Article Snippet: Cytokines, blocking TL1A antibody (TL1AAb) and Cyclosporine A (CsA) were added in the following concentrations: IL-12 (RnD Systems, Cat# 219-IL): 4 ng/mL, IL-15 (Peprotech, Cat# 200-15): 10 ng/mL, IL-18 (MBL, Cat# B003-5): 40 ng/mL, TL1A (RnD Systems, Cat# 1319-TL): 100 ng/mL, TL1AAb (RnD Systems, Cat# MAB7441): 1 μg/mL, CsA (Sigma-Aldrich, Cat# C1832):1 μg/mL.

Techniques: Light Microscopy

Journal: PLoS ONE

Article Title: TL1A Increases Expression of CD25, LFA-1, CD134 and CD154, and Induces IL-22 and GM-CSF Production from Effector CD4 T-Cells

doi: 10.1371/journal.pone.0105627

Figure Lengend Snippet: Purified PBLs were stimulated for 12 days with CD3/CD28 beads. After 12 days, beads were removed and the cells set up in new media. Cytokines were added in the following concentrations IL-12: 4 ng/mL, IL-15: 10 ng/mL, IL-18: 40 ng/mL, TL1A: 100 ng/mL, TL1A Ab: 1 µg/mL. Surface expression of co-receptors and activation markers CD25, CD134, CD154 and LFA-1 after 72 h. Upper panels: Gating strategy for lymphocytes. Lower panels: Co-staining for CD4 and CD134, CD154, CD25 of LFA-1, % positive cells and MFI for upper right quadrant are shown. Data are representative of results obtained with cells from three different donors.

Article Snippet: Cytokines, blocking TL1A antibody (TL1AAb) and Cyclosporine A (CsA) were added in the following concentrations: IL-12 (RnD Systems, Cat# 219-IL): 4 ng/mL, IL-15 (Peprotech, Cat# 200-15): 10 ng/mL, IL-18 (MBL, Cat# B003-5): 40 ng/mL, TL1A (RnD Systems, Cat# 1319-TL): 100 ng/mL, TL1AAb (RnD Systems, Cat# MAB7441): 1 μg/mL, CsA (Sigma-Aldrich, Cat# C1832):1 μg/mL.

Techniques: Purification, Expressing, Activation Assay, Staining

Journal: PLoS ONE

Article Title: TL1A Increases Expression of CD25, LFA-1, CD134 and CD154, and Induces IL-22 and GM-CSF Production from Effector CD4 T-Cells

doi: 10.1371/journal.pone.0105627

Figure Lengend Snippet: Purified PBLs were stimulated for 12 days with CD3/CD28 beads. After 12 days, beads were removed and the cells set up in new media. Cytokines/inhibitors were added in the following concentrations IL-12: 4 ng/mL, IL-15: 10 ng/mL, IL-18: 40 ng/mL, TL1A: 100 ng/mL, TL1A Ab: 1 µg/mL, CsA 1 µg/mL. Surface markers were measured at t = 6, 24, 72, 96, 168 and 240 h. Cells/µL was multiplied by MFI (cells/µL*MFI) to take both expression levels and cell growth into account. Data are representative of results obtained with cells from three different donors.

Article Snippet: Cytokines, blocking TL1A antibody (TL1AAb) and Cyclosporine A (CsA) were added in the following concentrations: IL-12 (RnD Systems, Cat# 219-IL): 4 ng/mL, IL-15 (Peprotech, Cat# 200-15): 10 ng/mL, IL-18 (MBL, Cat# B003-5): 40 ng/mL, TL1A (RnD Systems, Cat# 1319-TL): 100 ng/mL, TL1AAb (RnD Systems, Cat# MAB7441): 1 μg/mL, CsA (Sigma-Aldrich, Cat# C1832):1 μg/mL.

Techniques: Purification, Expressing

Journal: PLoS ONE

Article Title: TL1A Increases Expression of CD25, LFA-1, CD134 and CD154, and Induces IL-22 and GM-CSF Production from Effector CD4 T-Cells

doi: 10.1371/journal.pone.0105627

Figure Lengend Snippet: A. Purified PBLs were stimulated for 12 days with CD3/CD28 beads, after which cells were stimulated with combinations of cytokines. Cytokines/inhibitors were added in the following concentrations: IL-12: 4 ng/mL, IL-15: 10 ng/mL, IL-18: 40 ng/mL, TL1A: 100 ng/mL, TL1A Ab: 1 µg/mL, CsA: 1 µg/mL Expression of surface markers was measured by flow cytometry after 96 h. Cells/µL was multiplied by MFI (cells/µL*MFI) to take both expression levels and cell growth into account. B. Freshly purified PBLs were stimulated with Cytokines/inhibitors in the following concentrations: IL-12: 4 ng/mL, IL-15: 10 ng/mL, IL-18: 40 ng/mL, TL1A: 100 ng/mL, TL1A Ab: 1 µg/mL, CsA: 1 µg/mL. Expression of surface markers was measured by flow cytometry after 72 h. Please note that the Y-axis of the graphs differ in range. Data are representative of results obtained with cells from three different donors.

Article Snippet: Cytokines, blocking TL1A antibody (TL1AAb) and Cyclosporine A (CsA) were added in the following concentrations: IL-12 (RnD Systems, Cat# 219-IL): 4 ng/mL, IL-15 (Peprotech, Cat# 200-15): 10 ng/mL, IL-18 (MBL, Cat# B003-5): 40 ng/mL, TL1A (RnD Systems, Cat# 1319-TL): 100 ng/mL, TL1AAb (RnD Systems, Cat# MAB7441): 1 μg/mL, CsA (Sigma-Aldrich, Cat# C1832):1 μg/mL.

Techniques: Purification, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: TL1A Increases Expression of CD25, LFA-1, CD134 and CD154, and Induces IL-22 and GM-CSF Production from Effector CD4 T-Cells

doi: 10.1371/journal.pone.0105627

Figure Lengend Snippet: Purified PBLs were stimulated for 12 days with CD3/CD28 beads. After 12 days, beads were removed and the cells set up in new media. Cytokines/inhibitors were added in the following concentrations IL-12: 4 ng/mL, IL-15: 10 ng/mL, IL-18: 40 ng/mL, TL1A: 100 ng/mL, TL1A Ab: 1 µg/mL, CsA 1 µg/mL Supernatant was collected 96 h after stimulation and cytokines measured by ELISA or multiplex as described in Materials and Methods. Mean +/− SEM on two measurements is shown. The addition of TL1A significantly increased the expression of cytokines compared to all other cytokine combinations tested;α IL-22 (P<0.0002) and GM-CSF (P<0.00005), t-test. Data are representative of results obtained with cells from three different donors.

Article Snippet: Cytokines, blocking TL1A antibody (TL1AAb) and Cyclosporine A (CsA) were added in the following concentrations: IL-12 (RnD Systems, Cat# 219-IL): 4 ng/mL, IL-15 (Peprotech, Cat# 200-15): 10 ng/mL, IL-18 (MBL, Cat# B003-5): 40 ng/mL, TL1A (RnD Systems, Cat# 1319-TL): 100 ng/mL, TL1AAb (RnD Systems, Cat# MAB7441): 1 μg/mL, CsA (Sigma-Aldrich, Cat# C1832):1 μg/mL.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Expressing

Journal: PLoS ONE

Article Title: TL1A Increases Expression of CD25, LFA-1, CD134 and CD154, and Induces IL-22 and GM-CSF Production from Effector CD4 T-Cells

doi: 10.1371/journal.pone.0105627

Figure Lengend Snippet: Purified PBLs were stimulated for 12 days with CD3/CD28 beads. After 12 days, beads were removed and the cells set up as PBLs or CD8 depleted PBLs in new media. A. Left panel: Gating strategy for lymphocytes. Middle panel: Single cells. Right panel: Verification of CD8 depletion by staining for CD4/CD3 (∼95% were CD3 + CD4 + ). B. IL-22 and GM-CSF production by PBLs and CD8 depleted cells from A. Supernatant was collected seven days after stimulation and cytokines measured by ELISA as described in Materials and Methods. Cytokines/inhibitors were added in the following concentrations IL-12: 4 ng/mL, IL-15: 10 ng/mL, IL-18: 40 ng/mL, TL1A: 100 ng/mL. Mean +/− SEM on two measurements is shown. Data are representative of results obtained with cells from two different donors.

Article Snippet: Cytokines, blocking TL1A antibody (TL1AAb) and Cyclosporine A (CsA) were added in the following concentrations: IL-12 (RnD Systems, Cat# 219-IL): 4 ng/mL, IL-15 (Peprotech, Cat# 200-15): 10 ng/mL, IL-18 (MBL, Cat# B003-5): 40 ng/mL, TL1A (RnD Systems, Cat# 1319-TL): 100 ng/mL, TL1AAb (RnD Systems, Cat# MAB7441): 1 μg/mL, CsA (Sigma-Aldrich, Cat# C1832):1 μg/mL.

Techniques: Purification, Staining, Enzyme-linked Immunosorbent Assay

Journal: PLOS One

Article Title: TL1A serves as a positive regulator to promote adipocyte differentiation

doi: 10.1371/journal.pone.0343036

Figure Lengend Snippet: Two days post-confluent MEFs or 3T3-L1 cells were cultured in adipogenic induction medium supplemented with TL1A recombinant protein at the specified concentrations (0, 10, 20, 50, 100, 200 ng/mL). (A) Oil Red O staining was conducted after adipogenic induction for 7 days. (B) The average optical density (OD) was measured using a Universal Microplate Spectrophotometer at 490 nm. (C) Measurement of triglyceride (TG) content in MEFs 7 days after adipogenic differentiation. (D-E) Both Oil Red O staining and triglyceride assays in 3T3-L1 cells were performed on day 7 of differentiation. (F-G) qRT-PCR analysis was conducted to evaluate the expression of adipogenic markers aP2, Plin1, Plin2 and PPARγ in 3T3-L1 cells 7 days after adipogenic differentiation. (H) qRT-PCR analysis was conducted to evaluate the expression of VEGFA and several adipogenic markers C/EBPα/β/δ, PPARγ2, aP2, CD36, Plin1, Plin2, adispin, Glut4 and LPL in MEFs 7 days after adipogenic differentiation. * P < 0.05, ** P < 0.01, *** P < 0.001 vs . the group of adipocytes without TL1A treatment (n = 3).

Article Snippet: Human TL1A recombinant protein (Cat# 1319-TL-010/CF) was purchased from R&D Systems.

Techniques: Cell Culture, Recombinant, Staining, Spectrophotometry, Quantitative RT-PCR, Expressing

Journal: PLOS One

Article Title: TL1A serves as a positive regulator to promote adipocyte differentiation

doi: 10.1371/journal.pone.0343036

Figure Lengend Snippet: (A-D) MEFs or 3T3-L1 cells were induced to undergo adipocyte differentiation for 3 days in the presence of the specified concentrations of TL1A. Total protein was extracted to determine C/EBPα, C/EBPβ, PPARγ2, PPARγ1 and aP2 protein expression by Western blotting with quantitation of band density. (E-H) MEFs or 3T3-L1 cells were induced to undergo adipocyte differentiation for 7 days with the indicated concentrations of TL1A. Total protein was extracted to determine PPARγ2, PPARγ1, CD36 and aP2 protein expression by Western blottingwith quantitation of band density. * P < 0.05, ** P < 0.01, *** P < 0.001 vs . the group of adipocytes without TL1A treatment (n = 3).

Article Snippet: Human TL1A recombinant protein (Cat# 1319-TL-010/CF) was purchased from R&D Systems.

Techniques: Expressing, Western Blot, Quantitation Assay

Journal: PLOS One

Article Title: TL1A serves as a positive regulator to promote adipocyte differentiation

doi: 10.1371/journal.pone.0343036

Figure Lengend Snippet: After two days post-confluence, MEFs were treated with standard differentiation medium and TL1A recombinant protein (0, 10, 20, 50, 100, 200 ng/mL). At specified time points, cells were harvested for RNA isolation and subsequent qRT-PCR analysis. * P < 0.05 vs . the group of adipocytes without TL1A treatment.

Article Snippet: Human TL1A recombinant protein (Cat# 1319-TL-010/CF) was purchased from R&D Systems.

Techniques: Recombinant, Isolation, Quantitative RT-PCR

Journal: PLOS One

Article Title: TL1A serves as a positive regulator to promote adipocyte differentiation

doi: 10.1371/journal.pone.0343036

Figure Lengend Snippet: MEFs were treated with standard differentiation medium and TL1A at the specified concentrations (0, 10, 20, 50, 100, 200 ng/mL) for 7 days. (A) Total cellular proteins were extracted, and the expression levels of β-catenin, phosphorylated YAP1 S127 (p-YAP1 S127 ) and total YAP1 proteins were determined by Western blotting. (B) The relative amounts of β-catenin protein expression were calculated according to the grayscale values and were showed in the histogram. (C) The quantitation of ratio (p-YAP1 S127 /total YAP1) to reflect the inactivation of YAP1 signaling pathway. (D-E) At the end of adipogenesis induction, protein expression of phosphorylated β-catenin (p- β-catenin) and YAP1 in the cytoplasm extract from MEFs was determined by Western blotting. (F-G) Nuclear extracts were isolated from the MEFs and analyzed for protein expression by Western blotting. Nuclear proteins were isolated to evaluated the expression of β-catenin, YAP1 and PPARγ proteins through Western blotting, accompanied by a quantitative analysis of band density. GAPDH and Lamin A/C served as the internal controls for cytoplasmic and nuclear extract, respectively. All the histograms represent the relative expression levels of proteins normalized by GAPDH or Lamin A/C. * P <0.05, ** P <0.01, *** P <0.001 vs . the group of MEFs without TL1A treatment.

Article Snippet: Human TL1A recombinant protein (Cat# 1319-TL-010/CF) was purchased from R&D Systems.

Techniques: Expressing, Western Blot, Quantitation Assay, Isolation

Journal: PLOS One

Article Title: TL1A serves as a positive regulator to promote adipocyte differentiation

doi: 10.1371/journal.pone.0343036

Figure Lengend Snippet: Exogenous TL1A triggers the phosphorylation of YAP1 at Ser127, resulting in its cytoplasmic retention and functional inactivation. This event subsequently reduces β-catenin stability and prevents its nuclear translocation, thereby triggering the expression of essential adipogenic regulators and lipid metabolism-related proteins. Additionally, TL1A-mediated regulation of ABCA1 and ABCG1 expression facilitates cholesterol homeostasis, providing essential substrates for lipid droplet biogenesis. Collectively, these molecular mechanisms orchestrate the differentiation of MEFs and 3T3-L1 preadipocytes into terminally differentiated adipocytes.

Article Snippet: Human TL1A recombinant protein (Cat# 1319-TL-010/CF) was purchased from R&D Systems.

Techniques: Phospho-proteomics, Functional Assay, Translocation Assay, Expressing