Journal: International journal of molecular sciences

Article Title: Lumican, an Exerkine, Protects against Skeletal Muscle Loss.

doi: 10.3390/ijms231710031

Figure Lengend Snippet: Figure 6. Lumican-stimulated myogenesis and protein balance mediated by integrins. (A) mRNA levels of integrin subunits in C2C12 cells were determined by real-time PCR. (B) C2C12 cells were differentiated with 10 nM lumican in the presence or absence of integrin α7 neutralizing antibody for 3 days. Myotubes were stained with the anti-myosin heavy chain antibody. Quantitative results of myotube area per myotube are shown. (C) C2C12 cells were differentiated with 10 nM lumican in the presence or absence of integrin inhibitors, such as TC-I-15 and echistatin, for 3 days. Quantitative results of myotube area per myotube are shown. (D) C2C12 cells were pre-treated with or without integrin inhibitors for 30 min. Then, cells were treated with 10 nM lumican for 30 min and underwent lysis after 30 min of incubation with puromycin. Protein synthesis was determined by detecting puromycin-labeled peptides by western blotting. Quantitative results are shown in the right panel. (E) Protein degradation was measured in C2C12 myoblasts cultured with lumican and/or integrin inhibitors, as described in Figure 3. Each bar represents the mean ± standard error of the mean (SEM). * p < 0.05 vs. lumican-untreated control. NS, not significant.

Article Snippet: To block signal transduction pathways, cells were pre-treated with SB203580 (p38 inhibitor, Sigma-Aldrich, St. Louis, MO, USA), TC-I-15 (α2β1 integrin inhibitor, R&D Systems), echistatin (ανβ3 integrin inhibitor, R&D Systems), or integrin α7 neutralizing antibody (Origene, Rockville, MD, USA).

Techniques: Real-time Polymerase Chain Reaction, Staining, Lysis, Incubation, Labeling, Western Blot, Cell Culture, Control

Journal: Transplantation

Article Title: Differential Regulation of T Cell Immunity and Tolerance by Stromal Laminin Expressed in the Lymph Node

doi: 10.1097/TP.0000000000002774

Figure Lengend Snippet: Stromal laminin 411 and 511 differentially modulate CD4+ T cell activation in mice and human. Murine CD4 T lymphocytes evaluated by flow cytometry for proliferation and CD69, CD25, CD44 expression (A, B) after 3 days of culture in plates coated with anti-CD3 (1 μg/mL) and the indicated concentrations of laminin 411 or laminin 511 (A), or with laminin 411 (2 μg/mL) and/or laminin 511 (1 μg/mL) (B). Human CD4+ T lymphocytes (C) evaluated by flow cytometry for proliferation and CD25 expression after 3 days of culture in plates coated with laminin 411 (2 μg/mL) and/or laminin 511 (1 μg/mL) with anti-CD3/CD28 beads (T:Beads ratio = 1:1). Data shown as representative profiles (B, C) and mean ± SEM (A-C) of three independent experiments with samples.

Article Snippet: *P < 0.05, **P < 0.01, ***P < 0.001. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Target Molecule Source Clone Company α1 integrin Hamster Ha31/8 BD Bioscience α2 integrin Armenian Hamster HMα2 Biolegend α3 integrin Mouse ASC-1 Biolegend α4 integrin Rat 9C10 Biolegend α5 integrin Armenian Hamster HMα5–1 Biolegend α6 integrin Rat GoH3 Biolegend α7 integrin Mouse 6A11 LSBio αM integrin Rat M1/70 Biolegend αV integrin Rat RMV-7 Biolegend β1 integrin Armenian Hamster HMβ1–1 Biolegend β2 integrin Rat M18/2 Biolegend β3 integrin Armenian Hamster 2C9.G2 Biolegend α dystroglycan Mouse IIH6C4 Merck Millipore Open in a separate window Potential anti-laminin receptor antibodies for in vitro use We next determined if the laminin 511 receptor α6 integrin favored CD4+ T cell activation in vivo.

Techniques: Activation Assay, Flow Cytometry, Expressing

Journal: Transplantation

Article Title: Differential Regulation of T Cell Immunity and Tolerance by Stromal Laminin Expressed in the Lymph Node

doi: 10.1097/TP.0000000000002774

Figure Lengend Snippet: Laminin 511 favors CD4 T cell activation via the receptor α6 integrin. CD4 T lymphocytes evaluated by flow cytometry for proliferation and CD69, CD25, CD44 expression (A), for α6 integrin expression after 3 days of culture (B), or phospho-Erk1/2, phospho-Akt and phospho-P38Mapk (C) after 30 minutes of culture in plates coated with anti-CD3 (1 μg/mL) and with laminin 411 (2 μg/mL) and/or laminin 511 (1 μg/mL) in presence of anti-α6 integrin or isotype control mAb (A, D). Data shown as representative profiles (B, C) and mean ± SEM (A-D) of three independent experiments with duplicate samples. (E) DST or (F) CFA/Ea treated mice received 5 × 106 CFSE-labeled CD45.1 TEa TCR transgenic CD4+ T cells. Mice were euthanized two days post transfer, and donor lymphocytes from spleen and skin LNs assessed for proliferation, CD69, CD25, CD44 and percentage of donor cells (CD4+ CD45.1+) in different organs by flow cytometry. Representative profiles shown together with a quantitative analysis of three independent experiments. Data shown as mean ± SEM of at least six mice per group from three independent experiments.

Article Snippet: *P < 0.05, **P < 0.01, ***P < 0.001. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Target Molecule Source Clone Company α1 integrin Hamster Ha31/8 BD Bioscience α2 integrin Armenian Hamster HMα2 Biolegend α3 integrin Mouse ASC-1 Biolegend α4 integrin Rat 9C10 Biolegend α5 integrin Armenian Hamster HMα5–1 Biolegend α6 integrin Rat GoH3 Biolegend α7 integrin Mouse 6A11 LSBio αM integrin Rat M1/70 Biolegend αV integrin Rat RMV-7 Biolegend β1 integrin Armenian Hamster HMβ1–1 Biolegend β2 integrin Rat M18/2 Biolegend β3 integrin Armenian Hamster 2C9.G2 Biolegend α dystroglycan Mouse IIH6C4 Merck Millipore Open in a separate window Potential anti-laminin receptor antibodies for in vitro use We next determined if the laminin 511 receptor α6 integrin favored CD4+ T cell activation in vivo.

Techniques: Activation Assay, Flow Cytometry, Expressing, Labeling, Transgenic Assay

Journal: Transplantation

Article Title: Differential Regulation of T Cell Immunity and Tolerance by Stromal Laminin Expressed in the Lymph Node

doi: 10.1097/TP.0000000000002774

Figure Lengend Snippet: Laminin 411 and 511 differentially regulate Th1 and Th2 polarization. CD4 T lymphocytes evaluated by flow cytometry for intracellular expression of IFNγ (A) or IL4 (D) after 5 days or Tbet, Gata3 and phospho-Stat6 after 16 hours culture in pro-Th1 (B,C) or pro-Th2 (E,F) environments with laminin 411 (2 μg/mL) and/or laminin 511 (1 μg/mL) in presence of functional blocking anti-α6 integrin or isotype control mAb (C,F). Data shown as representative profiles and mean ± SEM of at least three independent experiments with duplicate samples.

Article Snippet: *P < 0.05, **P < 0.01, ***P < 0.001. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Target Molecule Source Clone Company α1 integrin Hamster Ha31/8 BD Bioscience α2 integrin Armenian Hamster HMα2 Biolegend α3 integrin Mouse ASC-1 Biolegend α4 integrin Rat 9C10 Biolegend α5 integrin Armenian Hamster HMα5–1 Biolegend α6 integrin Rat GoH3 Biolegend α7 integrin Mouse 6A11 LSBio αM integrin Rat M1/70 Biolegend αV integrin Rat RMV-7 Biolegend β1 integrin Armenian Hamster HMβ1–1 Biolegend β2 integrin Rat M18/2 Biolegend β3 integrin Armenian Hamster 2C9.G2 Biolegend α dystroglycan Mouse IIH6C4 Merck Millipore Open in a separate window Potential anti-laminin receptor antibodies for in vitro use We next determined if the laminin 511 receptor α6 integrin favored CD4+ T cell activation in vivo.

Techniques: Flow Cytometry, Expressing, Functional Assay, Blocking Assay

Journal: Transplantation

Article Title: Differential Regulation of T Cell Immunity and Tolerance by Stromal Laminin Expressed in the Lymph Node

doi: 10.1097/TP.0000000000002774

Figure Lengend Snippet: Laminin 511 promotes Th17 polarization via the α-dystroglycan receptor. CD4 T lymphocytes evaluated by flow cytometry for IL-17 after 5 days of culture (A, C), Rorγt, phopho-Stat3, Irf4, Batf and CD126 (B, D) expression after 16 hours of culture in pro-Th17 environment with laminin 411 (2 μg/mL) and/or laminin 511 (1 μg/mL) in presence of functional blocking anti-α6 integrin mAb, anti-α-dystroglycan or isotype control (C, D). Data shown as representative profiles (A, B) and mean ± AEM (A-D) of at least three independent experiments with duplicate samples. (E) CFA/Ea treated mice received 5 × 106 CFSE-labeled CD45.1 Ea TCR transgenic CD4+ T cells, with or without anti-αDG mAb, euthanized three days post transfer, and donor lymphocytes from spleen and skin LNs assessed as percentage of IL17+ donor cells (CD4+ CD45.1+) in different organs by flow cytometry. Representative profile shown with quantitative analysis of three independent experiments. Data shown as mean ± SEM of eight mice per group from three independent experiments.

Article Snippet: *P < 0.05, **P < 0.01, ***P < 0.001. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Target Molecule Source Clone Company α1 integrin Hamster Ha31/8 BD Bioscience α2 integrin Armenian Hamster HMα2 Biolegend α3 integrin Mouse ASC-1 Biolegend α4 integrin Rat 9C10 Biolegend α5 integrin Armenian Hamster HMα5–1 Biolegend α6 integrin Rat GoH3 Biolegend α7 integrin Mouse 6A11 LSBio αM integrin Rat M1/70 Biolegend αV integrin Rat RMV-7 Biolegend β1 integrin Armenian Hamster HMβ1–1 Biolegend β2 integrin Rat M18/2 Biolegend β3 integrin Armenian Hamster 2C9.G2 Biolegend α dystroglycan Mouse IIH6C4 Merck Millipore Open in a separate window Potential anti-laminin receptor antibodies for in vitro use We next determined if the laminin 511 receptor α6 integrin favored CD4+ T cell activation in vivo.

Techniques: Flow Cytometry, Expressing, Functional Assay, Blocking Assay, Labeling, Transgenic Assay

Journal: Transplantation

Article Title: Differential Regulation of T Cell Immunity and Tolerance by Stromal Laminin Expressed in the Lymph Node

doi: 10.1097/TP.0000000000002774

Figure Lengend Snippet: Laminin 411 and 511 differentially regulate Treg polarization. CD4 T lymphocytes evaluated by flow cytometry for CD25, FoxP3 expression after 5 days of culture (A, C), Rorγt, phopho-Stat3, Irf4 and Batf (B, D) expression after 16 hours of culture in pro-Treg environment, with laminin 411 (2 μg/mL) and/or laminin 511 (1 μg/mL) in presence of functional blocking anti-α6 integrin or isotype control mAb (C, D). Data shown as representative profiles (A-C) and mean ± SEM (A-D) of at least three independent experiments with duplicate samples.

Article Snippet: *P < 0.05, **P < 0.01, ***P < 0.001. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Target Molecule Source Clone Company α1 integrin Hamster Ha31/8 BD Bioscience α2 integrin Armenian Hamster HMα2 Biolegend α3 integrin Mouse ASC-1 Biolegend α4 integrin Rat 9C10 Biolegend α5 integrin Armenian Hamster HMα5–1 Biolegend α6 integrin Rat GoH3 Biolegend α7 integrin Mouse 6A11 LSBio αM integrin Rat M1/70 Biolegend αV integrin Rat RMV-7 Biolegend β1 integrin Armenian Hamster HMβ1–1 Biolegend β2 integrin Rat M18/2 Biolegend β3 integrin Armenian Hamster 2C9.G2 Biolegend α dystroglycan Mouse IIH6C4 Merck Millipore Open in a separate window Potential anti-laminin receptor antibodies for in vitro use We next determined if the laminin 511 receptor α6 integrin favored CD4+ T cell activation in vivo.

Techniques: Flow Cytometry, Expressing, Functional Assay, Blocking Assay

Journal: Transplantation

Article Title: Differential Regulation of T Cell Immunity and Tolerance by Stromal Laminin Expressed in the Lymph Node

doi: 10.1097/TP.0000000000002774

Figure Lengend Snippet: Blocking laminin 511 receptor α6 integrin and αDG prolongs allogenic cardiac graft survival. C57BL/6 mice received BALB/c heterotopic cardiac allografts on day 0 along with anti-α6 integrin or isotype control mAb alone (10 μg i.v, day 0 and every 3 days) (A), anti- αDG or isotype control mAb alone (B), anti-α6 integrin or anti- αDG (10 μg i.v, day 0 and once a week) with anti-CD40L (250 μg i.v. day 0 only) (C). Data presented as percent survival. n = at least five mice per group.

Article Snippet: *P < 0.05, **P < 0.01, ***P < 0.001. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Target Molecule Source Clone Company α1 integrin Hamster Ha31/8 BD Bioscience α2 integrin Armenian Hamster HMα2 Biolegend α3 integrin Mouse ASC-1 Biolegend α4 integrin Rat 9C10 Biolegend α5 integrin Armenian Hamster HMα5–1 Biolegend α6 integrin Rat GoH3 Biolegend α7 integrin Mouse 6A11 LSBio αM integrin Rat M1/70 Biolegend αV integrin Rat RMV-7 Biolegend β1 integrin Armenian Hamster HMβ1–1 Biolegend β2 integrin Rat M18/2 Biolegend β3 integrin Armenian Hamster 2C9.G2 Biolegend α dystroglycan Mouse IIH6C4 Merck Millipore Open in a separate window Potential anti-laminin receptor antibodies for in vitro use We next determined if the laminin 511 receptor α6 integrin favored CD4+ T cell activation in vivo.

Techniques: Blocking Assay