Journal: Nature communications

Article Title: Loss of vascular endothelial notch signaling promotes spontaneous formation of tertiary lymphoid structures.

doi: 10.1038/s41467-022-29701-x

Figure Lengend Snippet: Fig. 2 Molecular, cellular and structural composition of periarterial TLS. A–I Immunofluorescence staining and confocal laser scanning microscopy of representative RbpjΔEC kidney samples, merged and single channels as indicated. “A” indicates artery. Optical Magnification 200x; different scan areas (see scale bars). Scale: solid bar 50 µm, dotted bar 10 µm (2E). Each micrograph is representative of at least 4 biological replicates. J Whole kidney mRNA expression, relative fold change to control gene Rps9, n = 10/group. Graphs: Scatter dot blot, mean, SD (whiskers). Mann–Whitney test, two-tailed, Exact p-values: Cxcl13, p = 0.0003; Cxcl12, p = 0.393; Cxcr5, p = 0.0433; Cxcr4, p = 0.0288; Ccl19, p = 0.0052; Baff, p = 0.0007; Rankl, p = 0.0005. Source data are provided as a Source Data file.

Article Snippet: Imaging using light sheet microscopy; for CD31/B220, we used a LaVision BioTec Ultramicroscope (Imaging Center Essen), for B220/Prox1, Lyve1 a LaVision BioTec UltraMicroscope II at Hannover Medical School (different wavelength laser equipment).

Techniques: Staining, Confocal Laser Scanning Microscopy, Expressing, Control, Dot Blot, MANN-WHITNEY, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption *

doi: 10.1074/jbc.M113.494542

Figure Lengend Snippet: Siglec-15 and DAP12 form a complex in osteoclasts, and Siglec-15 clustering induces DAP12 phosphorylation and Akt signaling. A, co-immunoprecipitation (Co-IP) of DAP12 and Siglec-15. Protein lysates were prepared from non-differentiated RAW264.7 cells (control RAW) or RAW264.7-derived osteoclasts. Siglec-15 (left panels) and DAP12 (middle panels) immunoprecipitates as well as total lysates (right panels) were analyzed by Western blotting with Siglec-15 and DAP12 antibodies. The no lysate IPs were performed using fresh lysis buffer. B, analysis of cell signaling induced by Siglec-15 clustering. Control (C) or differentiated (D) RAW264.7 cells were treated with primary antibody (anti-Siglec-15 or control human IgG) at 4 °C followed by a secondary cross-linking antibody for the indicated times at 37 °C. Total lysates were analyzed by Western blotting with the indicated antibodies. C, DAP12 phosphorylation following Siglec-15 cross-linking. RAW264.7 cells were treated as in B for 5 min with secondary antibody (lanes 1, 2, and 5). As additional controls, some cells were lysed immediately after primary antibody incubation (lane 3) and for others, the secondary antibody was omitted from the 37 °C incubation media (lane 4). Protein extracts were prepared, and DAP12 immunoprecipitates were analyzed by blotting with anti-phosphotyrosine, anti-DAP12, and anti-Siglec-15 antibodies.

Article Snippet: Fab Preparation and Analysis Fab fragments were obtained from antibody B02 by ficin digest using the Mouse IgG1 Fab and F(ab′) 2 Preparation Kit (Pierce), according the manufacturer's instructions.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Derivative Assay, Western Blot, Lysis, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption *

doi: 10.1074/jbc.M113.494542

Figure Lengend Snippet: Antibody-induced internalization and lysosomal degradation of Siglec-15 in osteoclasts. A, internalization of biotinylated Siglec-15. RAW264.7-derived osteoclast cell-surface proteins were labeled with a disulfide-linked biotinylation reagent. Cells were then treated with a combination of primary (anti-Siglec-15 B02 or control IgG) antibodies (at 4 °C) followed by secondary cross-linking antibodies (10 min at 37 °C, lanes 5 and 6), or with primary antibody alone (10 min at 37 °C, lanes 7 and 8). Control cells were incubated with B02, control IgG, or no antibody at 4 °C (lanes 1–3) without a 37 °C chase. After these antibody treatments, remaining cell-surface biotin was stripped with a reducing agent; for one sample, as an additional control (lane 4), this stripping step was omitted. Internalized, biotinylated proteins were collected from cell lysates by streptavidin immunoprecipitation, and Siglec-15 was detected by Western blotting. B, characterization of Siglec-15 endocytosis by confocal microscopy. RAW264.7-derived osteoclasts were cold-loaded with Siglec-15 antibody and either fixed immediately (No chase) or incubated in fresh, warm media for 10 or 45 min prior to fixation. Cells were then permeablized and stained with anti-LAMP2 and anti-human IgG (to detect internalized Siglec-15). C, Siglec-15 protein levels decrease rapidly following antibody treatment. Differentiated RAW264.7-derived osteoclasts were treated for the indicated times with anti-Siglec-15 or a control human IgG. Protein lysates were analyzed by Western blotting with the indicated antibodies. D, RAW264.7 differentiated in the presence of Siglec-15 antibodies show decreased Siglec-15 protein levels. Protein lysates of RAW264.7 cells, either non-differentiated (− RANKL) or differentiated (+ RANKL) in media with or without anti-Siglec-15 were analyzed by Western blotting with the indicated antibodies.

Article Snippet: Fab Preparation and Analysis Fab fragments were obtained from antibody B02 by ficin digest using the Mouse IgG1 Fab and F(ab′) 2 Preparation Kit (Pierce), according the manufacturer's instructions.

Techniques: Derivative Assay, Labeling, Incubation, Stripping Membranes, Immunoprecipitation, Western Blot, Confocal Microscopy, Staining

Journal: The Journal of Biological Chemistry

Article Title: Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption *

doi: 10.1074/jbc.M113.494542

Figure Lengend Snippet: Siglec-15 antibodies inhibit osteoclast differentiation and activity in vitro. A, HOPs were grown with macrophage colony-stimulating factor alone (left panels) or with macrophage colony-stimulating factor and RANKL (to induce osteoclast differentiation) in the presence of control human IgG (middle panels) or anti-Siglec-15 (right panels) for 7 days. Cells were grown in parallel either on plastic (top panels) or on calcium phosphate-coated (bottom panels) plates. Multinucleated osteoclasts were identified on plastic by TRAP staining (red). Regions of calcium phosphate resorption were visualized by phase-contrast mode light microscopy after cell removal. B, upon prolonged treatment with RANKL (10 days rather than 7 days), HOPs grown in the presence of anti-Siglec-15 E09 fuse to form intensely TRAP-stained, multinucleated cells (right panel). The morphology of these cells is distinct from normal osteoclasts formed in the presence of a control antibody (middle panel). C, HOPs differentiated with RANKL in the presence of anti-Siglec-15 E09 display impaired TRAP secretion. TRAP levels in conditioned media of HOPs differentiated for 10 days were determined by ELISA. D, Siglec-15 antibody B02 inhibits differentiation of mouse RAW264.7 cells into osteoclasts. RAW264.7 cells were treated with RANKL to induce differentiation in the presence of control human IgG (middle panel) or anti-Siglec-15 B02 (right panel). Control cells were grown without RANKL or antibodies (left panel). After 3 days in culture, cells were stained for TRAP activity (red). The black arrows in the middle panel indicate examples of multinuclear, TRAP-positive osteoclasts.

Article Snippet: Fab Preparation and Analysis Fab fragments were obtained from antibody B02 by ficin digest using the Mouse IgG1 Fab and F(ab′) 2 Preparation Kit (Pierce), according the manufacturer's instructions.

Techniques: Activity Assay, In Vitro, Staining, Light Microscopy, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption *

doi: 10.1074/jbc.M113.494542

Figure Lengend Snippet: Treatment with Siglec-15 antibodies increases bone mineral density in mice. Three- to 4-week-old male mice were treated with the B02 Siglec-15 antibody twice per week at 1, 3, or 10 mg/kg for 4 weeks. A, bone mineral density of long bones (left panel, right femur; middle panel, right tibia) and the lumbar vertebrae (L4–L6, right panel) were measured with a densitometer. The control group was treated similarly with a murine IgG at 10 mg/kg. B, cross-sectional images of a representative right femur (top panels) dissected from a control IgG-treated mouse (control IgG, left panel) or a mouse treated with 10 mg/kg of the B02 Siglec-15 antibody (Anti-Siglec-15, right panel). Similar cross-sectional images of the L5 vertebra from the same mice are shown in the lower panels. C, osteoclast-specific TRAP (TRAP 5b) activity was measured by ELISA in serum prepared from a terminal bleed. p values (versus the IgG treated group, n = 5/group) were calculated using Student's t test. *, p < 0.05; **, p < 0.02.

Article Snippet: Fab Preparation and Analysis Fab fragments were obtained from antibody B02 by ficin digest using the Mouse IgG1 Fab and F(ab′) 2 Preparation Kit (Pierce), according the manufacturer's instructions.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption *

doi: 10.1074/jbc.M113.494542

Figure Lengend Snippet: Treatment with Siglec-15 antibody results in increased osteoclast number and osteoblast surface. A, representative photomicrographs (×10 magnification) of undecalcified mouse femur sections treated with control antibody (IgG) or anti-Siglec-15, histochemically stained with TRAP (A) and ALP (C). Scale bar, 100 μm. Histomorphometrical analysis was performed. B, N.Oc/BS, number of TRAP-positive osteoclasts per trabecular bone surface (mm). D, Ob.S/BS, ALP-positive osteoblast-surface per bone surface. p values (versus the IgG treated group, n = 5/group) were calculated using the Student's t test. *, p < 0.05.

Article Snippet: Fab Preparation and Analysis Fab fragments were obtained from antibody B02 by ficin digest using the Mouse IgG1 Fab and F(ab′) 2 Preparation Kit (Pierce), according the manufacturer's instructions.

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption *

doi: 10.1074/jbc.M113.494542

Figure Lengend Snippet: Monovalent Fab fragments have reduced ability to induce degradation of Siglec-15 or inhibit osteoclast differentiation. A, flow cytometry analysis of IgG/Fab binding to Siglec-15 expressed on intact cells. Full-length Siglec-15 was expressed in 2936E cells by transient transfection and cells were labeled with the indicated concentrations of anti-Siglec-15 B02 full IgG or Fab fragments. The values in the graph correspond to the mean fluorescence intensity of the transfected cells. B, RAW264.7-derived osteoclasts were treated with the indicated concentrations of anti-Siglec-15 B02 IgG or Fab or a control human IgG (10 μg/ml) for 2 h. Protein lysates were analyzed by Western blotting with the indicated antibodies. C, RAW264.7 cells were differentiated in the presence of the indicated IgG and Fab concentrations and stained for TRAP expression.

Article Snippet: Fab Preparation and Analysis Fab fragments were obtained from antibody B02 by ficin digest using the Mouse IgG1 Fab and F(ab′) 2 Preparation Kit (Pierce), according the manufacturer's instructions.

Techniques: Flow Cytometry, Binding Assay, Transfection, Labeling, Fluorescence, Derivative Assay, Western Blot, Staining, Expressing