Journal: The Journal of Clinical Investigation

Article Title: Nonimmune cell–derived ICOS ligand functions as a renoprotective αvβ3 integrin–selective antagonist

doi: 10.1172/JCI123386

Figure Lengend Snippet: (A) Schematic of a gold surface with ICOSL protein on a sensor chip CM5 and associated protein (αvβ3 integrin) over which buffer is flown in SPR assay. (B–I) SPR sensorgrams depicting interaction of immobilized human ICOSL (hICOSL, B–D) or mouse ICOSL (mICOSL, E–I) with αvβ3 integrin. These bindings were tested in the presence (B and E, active form of αvβ3) or absence (C and F, inactive form of one with EDTA in the binding buffer) of Mn2+. (D and G) SPR used in an inhibition experiment with cRGDfv. Injection of αvβ3 integrin only (D, 120 nM αvβ3 or G, 150 nM αvβ3) resulted in a binding signal for immobilized hICOSL or mICOSL alone (pink line). Preincubation with cRGDfv (3 μM or 15 μM) significantly reduced the binding for ICOSL, indicating that the RGD peptide competes with ICOSL for binding to αvβ3 (orange line). cRGDfv alone was used as a control (green line). (H and I) SPR sensorgrams showing the binding between WT (H) or mutant (I) mICOSL protein and αvβ3 integrin in the presence of physiologically relevant divalent ions, Ca2+ (0.2 mM) and Mg2+ (0.1 mM). The average KD values were determined from at least 3 independent experiments. Rate constants (ka and kd) were determined by kinetic fitting (black dotted line) of the sensorgrams using 1-to-1 Langmuir binding equation, and KD values for B, E, and H were calculated by kd/ka (B, KD = 16.2 ± 4.0 nM for hICOSL/αvβ3 with Mn2+; E, KD = 24.2 ± 6.5 nM for mICOSL/αvβ3 with Mn2+; H, KD = 21.3 ± 1.2 nM for WT mICOSL/αvβ3 with Ca2+/Mg2+). KD values for C, F, and I were calculated from steady-state affinity fittings (C, KD = 411.8 ± 164.1 nM for hICOSL/αvβ3; F, KD ≥ 2 mM for mICOSL/αvβ3; I, KD = 0.83 ± 0.8 mM for mutant mICOSL/αvβ3 with Ca2+/Mg2+).

Article Snippet: Recombinant proteins used in this study were as follows: human integrin αvβ3 (R&D Systems, 3050-AV), human integrin α3β1 (R&D Systems, 2840-A3), human integrin αIIbβ3 (R&D Systems, 7148-A2), human integrin αvβ5 (R&D Systems, 2528-AV), human fibronectin (R&D Systems, 4305-FNB-200), human ICOSL-His Tag (Thermo Fisher Scientific, 11559H08H50), mouse integrin αvβ3 (R&D Systems, 7889-AV-050), mouse ICOSL (Sino Biological, 50-190-M08H25), human B7.1 (R&D Systems, 140-B1), and human ICOS (R&D Systems, 169-CS).

Techniques: SPR Assay, Binding Assay, Inhibition, Injection, Control, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: Nonimmune cell–derived ICOS ligand functions as a renoprotective αvβ3 integrin–selective antagonist

doi: 10.1172/JCI123386

Figure Lengend Snippet: (A) Schematic representation of the protocol to measure relative cell adhesion levels in cultured human podocytes. Image analysis and quantification by high-content screening technology were described in Methods. (B) Phase-contrast microscopy images show that cultured human podocytes confer enhanced adhesion to ICOSL mediated by β3 integrin treated with Mn2+, but do not adhere on albumin (protein control). Increased adhesion levels were completely prevented by incubation with the integrin inhibitors, including cRGD peptide and anti-β3 integrin antibody. Scale bar 100 μm. (C) Quantification of the cell adhesion using the images in B. ICOSL induced cell adhesion to RGD-dependent β3 integrin on cultured podocytes. (D) Cell adhesion analysis of cultured podocytes plated on vitronectin. Data are shown as mean ± SD; ***P < 0.001; 1-way ANOVA with Tukey’s multiple comparison test (C and D).

Article Snippet: Recombinant proteins used in this study were as follows: human integrin αvβ3 (R&D Systems, 3050-AV), human integrin α3β1 (R&D Systems, 2840-A3), human integrin αIIbβ3 (R&D Systems, 7148-A2), human integrin αvβ5 (R&D Systems, 2528-AV), human fibronectin (R&D Systems, 4305-FNB-200), human ICOSL-His Tag (Thermo Fisher Scientific, 11559H08H50), mouse integrin αvβ3 (R&D Systems, 7889-AV-050), mouse ICOSL (Sino Biological, 50-190-M08H25), human B7.1 (R&D Systems, 140-B1), and human ICOS (R&D Systems, 169-CS).

Techniques: Cell Culture, High Content Screening, Microscopy, Control, Incubation, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Nonimmune cell–derived ICOS ligand functions as a renoprotective αvβ3 integrin–selective antagonist

doi: 10.1172/JCI123386

Figure Lengend Snippet: In this study, it is shown that ICOSL binds podocyte αvβ3 integrin through its RGD motif. Kidney injury results in a rapid increase of ICOSL expression, leading to podocyte protection by blocking active αvβ3 integrin. ICOSL acts as a regulatory brake to modulate active αvβ3 integrin–mediated signaling. Fp, foot process.

Article Snippet: Recombinant proteins used in this study were as follows: human integrin αvβ3 (R&D Systems, 3050-AV), human integrin α3β1 (R&D Systems, 2840-A3), human integrin αIIbβ3 (R&D Systems, 7148-A2), human integrin αvβ5 (R&D Systems, 2528-AV), human fibronectin (R&D Systems, 4305-FNB-200), human ICOSL-His Tag (Thermo Fisher Scientific, 11559H08H50), mouse integrin αvβ3 (R&D Systems, 7889-AV-050), mouse ICOSL (Sino Biological, 50-190-M08H25), human B7.1 (R&D Systems, 140-B1), and human ICOS (R&D Systems, 169-CS).

Techniques: Expressing, Blocking Assay

Journal: ACS Omega

Article Title: Design and Evaluation of Short Self-Assembling Depsipeptides as Bioactive and Biodegradable Hydrogels

doi: 10.1021/acsomega.7b01641

Figure Lengend Snippet: Half inhibition (i.e., IC 50 ) of the fluorescence of FITC-GRGDSP occurs at a ∼40-fold lower concentration for RGD than R-Glc-D, indicating greater affinity of RGD for integrin than R-Glc-D (A). Comparison of FP inhibition by R-Glc-D and RGE peptides (negative control). The average IC 50 value for R-Glc-D is 1115 ± 211 μM (mean ± standard deviation). RGE was tested once as a negative control, and the calculated IC 50 value was 2840 μM (B).

Article Snippet: Human recombinant integrin α 5 β 1 (R&D Systems) was diluted to 900 nM in the same Tris buffer.

Techniques: Inhibition, Fluorescence, Concentration Assay, Comparison, Negative Control, Standard Deviation