Journal: Bone Research

Article Title: Osteomodulin downregulation is associated with osteoarthritis development

doi: 10.1038/s41413-023-00286-5

Figure Lengend Snippet: OMD inhibits osteoclastogenesis through its direct interaction with RANKL and balances bone remodeling. a Solid phase binding assay on the capture of RANKL by OMD. RANKL was coated on a plate followed by OMD addition. On the left: Binding assay with different concentrations of OMD (1 000 to 15.65 ng·mL −1 by serial 2X dilution), with 0.2 μg·mL −1 of coated RANKL (red curve) and negative control without RANKL (blue curve). On the right: Binding assay with different concentrations of coated RANKL (800 to 6.25 ng·mL −1 ) and 0.5 μg·mL −1 of given OMD (pink curve); negative control without OMD (purple curve). Wilcoxon test was performed with differences being considered significant at P values < 0.05 (* P < 0.05, ** P ≤ 0.01). b Assay of the effect of OMD on primary murine osteoclast culture. The peripheral blood mononuclear cells were collected from murine bone marrow and differentiated into osteoclasts with M-CSF and RANKL. Osteoclasts were counted after 4 days of differentiation following a TRAP staining. Each point represents a mouse, n = 7. The osteoclast count was represented in percentage of cells with the corresponding control set as 100%. Blue arrows point at osteoclasts. Scale bar = 50 µm. The data were plotted as a box plot showing all points. One-way ANOVA was performed with differences being considered significant at P values <0.05 (* P < 0.05). c Level of P1NP and TRAcP 5b measured in the serum of KO, WT and UP mice. The data were plotted as a box plot showing all points with n = 12 for the KO and WT, and n = 10 for the UP. One-way ANOVA was performed when the distribution was Gaussian and Kruskal–Wallis was performed when the distribution was not Gaussian with differences being considered significant at P values < 0.05 (* P < 0.05, ** P ≤ 0.01, **** P ≤ 0.000 1). d Schematic representation of the mechanism of OMD on osteoclastogenesis. Osteoblasts secrete RANKL which binds to the RANK receptor on the membrane of pre-osteoclasts to induce their differentiation into osteoclasts. In parallel, osteoblasts also secrete OMD which displays the ability to capture RANKL and would prevent its binding to RANK

Article Snippet: Human recombinant RANKL (OriGene, Germany) was bound for 2 h under constant agitation to Well-CoatedTM Nickel (G-Biosciences) previously washed with PBST.

Techniques: Binding Assay, Negative Control, Staining, Control, Membrane

Journal: Medicine

Article Title: Determination of diagnostic and predictive parameters for vertical mandibular invasion in patients with lower gingival squamous cell carcinoma: A retrospective study

doi: 10.1097/MD.0000000000032206

Figure Lengend Snippet: Immunohistochemical staining.

Article Snippet: IL-6 , Rockland Inc. , 1:2000 , Rabbit polyclonal antibody , • Inflammatory cytokines released by cancer cells. • Since stimulation by IL-6 and TNF-α induces bone-resorbing multinucleated giant cells, it is possible that some mechanisms of the bone-resorptive effects of IL-6 are not mediated by the RANK/RANKL pathway..

Techniques: Immunohistochemical staining, Staining, Binding Assay, Activity Assay, Expressing

Journal:

Article Title: A TNF receptor loop peptide mimic blocks RANK ligand-induced signaling, bone resorption, and bone loss

doi: 10.1172/JCI22513

Figure Lengend Snippet: (A) Comparison of the sequence of peptide WP9QY with the corresponding sequences of domain 3 of TNFR(I), RANK, OPG, CD40, Fas, and death receptor 5 (DR5). The residues in the TNFR and RANK that are homologous to the peptide are underlined. OPG, CD40, Fas, and DR5 lack the sequence in loop 1 that is mimicked by WP9QY. (B) Space-filling model of the RANKL-WP9QY complex. Two RANKL monomers are shown (blue and white). The WP9QY atoms are colored green (carbon), red (oxygen), blue (nitrogen), and yellow (sulfur). Left: side view, with the complex oriented as if it were bound to RANK on the surface of a cell located below the complex; right: view of the complex rotated 90° with the bottom of the left image toward the viewer. The arrow indicates the location of Tyr6, which the modeling predicts to play an important role in WP9QY-RANKL binding. (C) Modeled complexes of RANK and RANKL extracellular domains with (right) and without (left) WP9QY. RANK and WP9QY are shown as space-filling models; RANKL is shown as a wire model. The complexes are viewed looking toward the RANK-expressing cell. The locations of the WP9QY peptides are indicated (arrowheads). Carbon atoms of RANK’s N-terminal and C-terminal domains are colored light blue and magenta, respectively. WP9QY and RANKL carbon atoms are colored yellow and green, respectively.

Article Snippet: Recombinant soluble human RANKL was from ProStrakan and Wako Pure Chemical Industries Inc. Recombinant human OPG was from PeproTech.

Techniques: Comparison, Sequencing, Binding Assay, Expressing

Journal:

Article Title: A TNF receptor loop peptide mimic blocks RANK ligand-induced signaling, bone resorption, and bone loss

doi: 10.1172/JCI22513

Figure Lengend Snippet: (A) The direct binding of sRANKL to the WP9QY peptide (filled circles) and to the Y6N control peptide (open circles) was examined by surface plasmon resonance, as described in Methods. Changing the Tyr residue to Asn reduced the binding by more than 70%. (B) The binding of RANKL (600 ng/ml) to the sRANK-coated surface was measured in the presence of increasing concentrations of WP9QY. Concentrations of WP9QY as high as 1 mM failed to significantly reduce the binding of sRANKL to RANK. (C) OPG effectively blocked the binding of sRANKL to RANK in a dose-dependent manner.

Article Snippet: Recombinant soluble human RANKL was from ProStrakan and Wako Pure Chemical Industries Inc. Recombinant human OPG was from PeproTech.

Techniques: Binding Assay, Control, SPR Assay, Residue

Journal:

Article Title: A TNF receptor loop peptide mimic blocks RANK ligand-induced signaling, bone resorption, and bone loss

doi: 10.1172/JCI22513

Figure Lengend Snippet: (A) RAW 264.7 cells treated with sRANKL with or without WP9QY were stained with anti-p65 antibody (green) and nuclear stain (blue). WP9QY prevented RANKL-induced nuclear accumulation of p65. (B) RAW 264.7 cells were treated with sRANKL with or without WP9QY (10 μM or 25 μM) or OPG. EMSA was performed using a consensus NF-κB oligonucleotide probe. WP9QY prevented the RANKL-induced activation of NF-κB. (C) Nonadherent bone marrow cells differentiated with M-CSF were treated for 48 hours with 100 ng/ml RANKL alone or with WP9QY (10 μM or 25 μM) or OPG. NFATc1 and c-Fos were detected by Western blotting. WP9QY inhibited RANKL-induced NFATc1 and c-Fos expression, albeit less effectively than OPG. (D) RAW 264.7 cells were treated with M-CSF (20 ng/ml for 5 minutes) or sRANKL, with or without 50 μM WP9QY as indicated. Phosphorylated and total Erk1/2 (upper panels) or phosphorylated and total glycogen synthase kinase 3α/β (GSK3α/β; lower panels) were assayed. WP9QY did not affect M-CSF–induced activation of Erk or Akt. (E) RAW 264.7 cells were treated with sRANKL with or without WP9QY (5–50 μM), the control Y6N peptide, or OPG as indicated. Erk, JNK, and Akt activation were monitored as described in Methods. Upper panels: phosphorylated and total Erk and phosphorylated and total c-Jun. The control peptide inhibited RANKL-induced activation of Erk1/2 and JNK significantly less than WP9QY. Lower panels: phosphorylated GSK3α/β. Protein loading was monitored by Ponceau S staining before blocking the membrane. WP9QY inhibited the RANKL-induced activation of Akt.

Article Snippet: Recombinant soluble human RANKL was from ProStrakan and Wako Pure Chemical Industries Inc. Recombinant human OPG was from PeproTech.

Techniques: Staining, Activation Assay, Western Blot, Expressing, Control, Blocking Assay, Membrane