antibody il34  (Bioss)

Journal: Theranostics

Article Title: Interleukin-34 orchestrates bone formation through its binding to bone morphogenic proteins

doi: 10.7150/thno.107340

Figure Lengend Snippet: Growth alterations associated with IL34 genetic invalidations in zebrafish and mouse . ( A ) Scheme of IL34 Exon3 genetic alterations induced by CrispR/Cas9 technology in zebrafish. ( B ) Images of zebrafish mutants compared to the control at age of 3 months. ( C ) Mineralization of craniofacial skeleton by Von Kossa and Alcian Blue staining of embryos at 5 days post fecundation. Abbreviations: mx - branchio maxilla, bs - branchistegal ray, op - opercle, cl - cleithrum, pt - pharyngeal teeth, m - Meckel's cartilage, pq - palatoquadrate, ch - ceratohyal, ep - ethmoid plate, marked 1-5 - different arches. ( D ) Scheme of Il34 floxed allele used to obtain constitutive invalidation of IL34 in mouse by removing exons 3 to 5 under CRE recombinase activity. ( E ) Images at 15 days after birth of consequences of the constitutive invalidation of IL34 with detail of hydrocephaly in Il34 -/- mouse (left panel). And comparative of skeletons at 15 postnatal days visualized by Alizarin red / Alcian blue double staining (right panel). ( F ) MicroCT scan 3D reconstructions of skull and tibia enable to visualize growth defects (red arrowheads). ( G ) Quantification of growth defects in the different morphometric planes (a to i) in wile type (black box) vs. Il34 -/- mice (red box), both treated with IK22.5 RANKL blocking antibody (blue and green boxes), or wile type mice with Sheff.5 IL34 blocking antibody (brown box). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The differences between the experimental conditions were assessed one-way ANOVA test. n=8 except for Il34 -/- + IK22.5 (n=4).

Article Snippet: Anti-human IL34 (BT-34) mouse IgG1 monoclonal antibody was produced by Diaclone (Besançon, France) under patent (Heymann D, Ségaliny A, Brion R. University of Nantes /Nantes Hospital/INSERM, “Anti-IL-34 antibodies”.

Techniques: CRISPR, Control, Staining, Activity Assay, Double Staining, Blocking Assay

Journal: Theranostics

Article Title: Interleukin-34 orchestrates bone formation through its binding to bone morphogenic proteins

doi: 10.7150/thno.107340

Figure Lengend Snippet: Bone mineral and histologic alterations associated with IL34 genetic invalidation in mouse. ( A ) Comparative analyses of skull bones mineralization levels between Il34 +/+ , Il34 -/- , Il34 +/+ injected with IK22.5 antibody, Il34 -/- injected with IK22.5 antibody and Il34 +/+ injected with Sheff.5 antibody mice at age of 15 days, using profile views of the microCT scan 3D reconstructions. The color density ranges from black (lower mineralization) to clear blue (higher mineralization). ( B ) Comparative analyses of tibias mineralization levels between Il34 +/+ , Il34 -/- , Il34 +/+ treated with IK22.5 antibody and Il34 -/- treated with IK22.5 antibody mice at age of 15 days, using longitudinal views of the microCT scan 3D reconstructions. ( C ) Comparative analysis of the bone volume (BV)/total volume (TV) ratio between Il34 +/+ , Il34 -/- , Il34 +/+ treated with IK22.5 antibody and Il34 -/- treated with IK22.5 antibody in bone of different anatomical sites: the mandible, the vertebra, the skull and the tibia. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. n=8 except for Il34 -/- + IK22.5 (n=4). ( D ) Comparative analysis of the bone mineral density (BMD) between Il34 +/+ , Il34 -/- , Il34 +/+ treated with IK22.5 antibody and Il34 -/- treated with IK22.5 antibody in bone of different anatomical sites: the mandible, the vertebra, the skull and the tibia. Two areas were considered for the tibia, the trabecular and the cortical. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. n=8 except for Il34 -/- + IK22.5 (n=4). ( E ) Chondrocytes stained by safranin-O staining of tibia longitudinal sections at the level of the proximal epiphysis performed for Il34 -/- and Il34 +/+ mice injected or not with the IK22.5 antibody. ( F ) Tartrate resistant acid phosphatase (TRAP) and Osterix (Osx/SP7) dual-staining of tibia longitudinal sections at the level of the proximal epiphysis performed for Il34 -/- and Il34 +/+ mice injected or not with the IK22.5 antibody. TRAP red staining for osteoclast cells. OSX brown staining for pre-osteoblasts cells. The scales are given as bars with the corresponding values in the lower part of each histological view.

Article Snippet: Anti-human IL34 (BT-34) mouse IgG1 monoclonal antibody was produced by Diaclone (Besançon, France) under patent (Heymann D, Ségaliny A, Brion R. University of Nantes /Nantes Hospital/INSERM, “Anti-IL-34 antibodies”.

Techniques: Injection, Staining

Journal: Theranostics

Article Title: Interleukin-34 orchestrates bone formation through its binding to bone morphogenic proteins

doi: 10.7150/thno.107340

Figure Lengend Snippet: IL34 regulates BMP2-associated osteoblastic and osteoclastic differentiation. ( A ) Images of human mesenchymal stem cells differentiated into osteoblasts cultured in basic culture medium (CT-) or in osteogenic culture medium (CT+) in the absence or presence of BMP2 (10 ng/mL), IL34 (20 ng/mL) or combination of both at 10 and 21 days. Right panel: quantification of alizarin red staining. Magnification was similar for all views and the bar in CT- view at day 10 corresponds to 500 µm. ( B ) Real-time PCR quantification of early ( RUNX2 ) and late ( ALP and OCN ) markers of osteoblastogenesis at days 0, 3 7 and 14. Data correspond to fold increase by 2 -ΔΔCt (cycle threshold) method. A representative experiment is shown. nd: non detected. ( C ) Western blot analysis of SMDA1/5 phosphorylation at different times of human mesenchymal stem cells differentiated into osteoblasts in basic culture medium (CT-) and in osteogenic culture medium (CT+) in the absence or presence of BMP2 (10 ng/mL), IL34 (20 ng/mL) or combination of both. ( D ) Differentiation of human CD14 + cells into osteoclastic cells analyzed by Tartrate Resistant Acid phosphatase activity (TRAP histoenzymology: purple staining) after 3-day culture period in the presence of MCSF (25 ng/mL) or IL34 (100 ng/mL), followed by an 8-day period of maturation with the addition of RANKL (100 ng/mL) and /or BMP2 addition (concentrations from 1 to 50 ng/mL) ( E ) Quantification of the different experiments repeated in triplicate and presented in D . At least two independent experiments have been carried in triplicate. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Anti-human IL34 (BT-34) mouse IgG1 monoclonal antibody was produced by Diaclone (Besançon, France) under patent (Heymann D, Ségaliny A, Brion R. University of Nantes /Nantes Hospital/INSERM, “Anti-IL-34 antibodies”.

Techniques: Cell Culture, Staining, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay

Journal: Theranostics

Article Title: Interleukin-34 orchestrates bone formation through its binding to bone morphogenic proteins

doi: 10.7150/thno.107340

Figure Lengend Snippet: The interaction IL34-BMP2 modulates SMAD1/5 as well as MCSF receptor (MCSFR) phosphorylation and related signaling. (A) Western blot analysis of SMDA1/5 and SMAD2 phosphorylations of human MNNG-HOS osteosarcoma cells in the presence of BMP2 (10 ng/mL), TGFß (10 ng/mL), MCSF (20 ng/mL), IL34 (20 ng/mL) alone or in corresponding combination. A representative experiment is shown. CT: basic culture medium. (B) Western blot analysis of SMDA1/5 phosphorylation of human MNNG-HOS osteosarcoma cells in the presence of BMP2 (10 ng/mL), IL34 (20 ng/mL) alone or in combination (BMP2+IL34, -) plus the human IL34 blocking antibody (BT34) (100 µg/mL) or its irrelevant isotypic control antibody (ISO) (100 µg/mL). CT: basic culture medium. (C) Western blot analysis of SMDA1/5, similar conditions used in B in the presence of the human IL34 blocking antibody (BT34) (100 µg/mL) or the natural inhibitor of BMP2 called NOGGIN (NOG) (200 ng/mL). (D) Kinetic analysis by Western blot of the potentiating effect of IL34 on BMP2-induced SMAD1/5 phosphorylation at 15 min, 30 min and 60 min with similar corresponding molecules concentrations described in B. (E) Western blot analysis of SMDA1/5 as described in B in the presence of a single concentration of BMP2 (10 ng/mL) in combination with gradual quantities of IL34 (10, 20, 40, 80 and 100 ng/mL). (F) The Alpha SureFire technology (Revvity) was used to quantitatively validate the potentiation effect of IL34 on BMP2 activation of SMAD1/5 phosphorylation. Co-additions of 25 or 50 ng/mL of IL34 increased significantly the phosphorylation of SMAD1/5 induced by the addition of BMP2 at 10 ng/mL. **p<0.01, ***p<0.001. (G) Western blot analysis of SMDA1/5 as described in B with a single concentration of IL34 (20 ng/mL) in combination with gradual quantities of IL34 (5, 10, 20, 40 and 80 ng/mL). (H) Western blot analysis of MCSFR phosphorylation expressed in HEK293 transfected cells in the presence or absence of BMP2 (10 ng/mL), IL34 (20 ng/mL) or in combination (BMP2+IL34, -) plus NOGGIN (NOG) (200 ng/mL). Quantifications of all the Western blots presented in this figure are shown in . All experiments have done at least three times independently.

Article Snippet: Anti-human IL34 (BT-34) mouse IgG1 monoclonal antibody was produced by Diaclone (Besançon, France) under patent (Heymann D, Ségaliny A, Brion R. University of Nantes /Nantes Hospital/INSERM, “Anti-IL-34 antibodies”.

Techniques: Western Blot, Blocking Assay, Control, Concentration Assay, Activation Assay, Transfection

Journal: Theranostics

Article Title: Interleukin-34 orchestrates bone formation through its binding to bone morphogenic proteins

doi: 10.7150/thno.107340

Figure Lengend Snippet: Demonstration and deciphering at the molecular level of the physical interaction between the IL34 protein and proteins of the BMP family. ( A ) Surface plasmon resonance experiments (described in Materials and Methods section) and values of proteins interaction parameters between IL34 and BMPs. ka: association rate constant, kd: dissociation rate constant, KD: affinity constant. ( B ) Molecular modelling of the binding of two BMPR1A receptors (green) to a BMP2 dimer (brown and dark blue) by using PyMOL. ( C ) Molecular modelling of the binding of two IL34 proteins (cyan) to a BMP2 dimer (brown and dark blue) seen in profile (top) and from above (bottom) with a representation of the BMP2 proteins in surface (left) and in structure (right) by using PyMOL. ( D ) Structural representation of a BMP2 dimer seen from above with the location of the “Knuckle” and “Wrist” binding sites to the type 1 and type 2 receptors respectively as described by Sebald and collaborators , . ( E ) Main amino acid of BMP2 and IL34 identified as being involved in binding. In addition, hydrogen bonds and salt bridges were found between BMP2 and IL34, more specifically between residues K383-D190, D312-K55 and E376-R73. ( F ) Localization on the representation of the BMP2 protein in structure of amino acids important for partner binding: F305 in red, W310 in bright green, W313 in yellow, Y385 in light brown and M388 in grey. These amino acids delineate the pocket in which residues F85 of BMPR1A and R48 of IL34 are positioned during their interaction with BMP2. The amino acid N341 presented in light blue, despite is localization in the most outside part of the pocket, was not identified as important for the binding to IL34. IL34 is displayed in surface representation with the entire binding region colored in yellow, and the important intercalating residue R48 is indicated in duck blue.

Article Snippet: Anti-human IL34 (BT-34) mouse IgG1 monoclonal antibody was produced by Diaclone (Besançon, France) under patent (Heymann D, Ségaliny A, Brion R. University of Nantes /Nantes Hospital/INSERM, “Anti-IL-34 antibodies”.

Techniques: SPR Assay, Binding Assay, Residue

Journal: Theranostics

Article Title: Interleukin-34 orchestrates bone formation through its binding to bone morphogenic proteins

doi: 10.7150/thno.107340

Figure Lengend Snippet: Impacts of the binding between BMP2 and IL34 on the ability of BMP2 and IL34 to bind to ACVR2A and MCSFR receptors respectively: importance of stoichiometry and functional consequences on bone formation and resorption. ( A ) ACVR2A receptor binding to BMP2 (“Wrist” site) does not appear to be affected by IL34 binding to the “Knuckle” sites of a BMP2 dimer. ( B ) MCSFR receptor binding to IL34 occurs at a site overlapping the BMP2 “Knuckle” site binding site. Simultaneous binding of BMP2 and MCSFR to IL34 is therefore impossible. ( C ) BMP receptor binding stoichiometry to a BMP2 dimer. The standard binding of two type 1 and two type 2 receptors per dimer (a), is gradually modified by the amount of IL34 present with potential transformation of a “Knuckle” site into a “Wrist”-like site at intermediate concentrations (b), then a second at high IL34 concentrations (c), bearing in mind that IL34 can bind type 2 BMP receptors. ( D ) Schematic representation of the impact of different ratios of BMPs and IL34 on bone formation and resorption.

Article Snippet: Anti-human IL34 (BT-34) mouse IgG1 monoclonal antibody was produced by Diaclone (Besançon, France) under patent (Heymann D, Ségaliny A, Brion R. University of Nantes /Nantes Hospital/INSERM, “Anti-IL-34 antibodies”.

Techniques: Binding Assay, Functional Assay, Modification, Transformation Assay