goat anti runx2  (R&D Systems)

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Bone-Targeted Delivery of Cell-Penetrating-RUNX2 Fusion Protein in Osteoporosis Model.

doi: 10.1002/advs.202301570

Figure Lengend Snippet: Figure 1. A schematic representation of the bone specific delivery of cell penetrable 30Kc19𝛼-RUNX2 protein for the treatment of osteoporosis. Created with BioRender.com.

Article Snippet: Anti-RUNX2 antibody (Santa Cruz Biotechnology, USA) was used as a primary antibody, and goat anti-mouse IgG-HRP antibody (AbFrontier, Korea) was used as a secondary antibody.

Techniques:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Bone-Targeted Delivery of Cell-Penetrating-RUNX2 Fusion Protein in Osteoporosis Model.

doi: 10.1002/advs.202301570

Figure Lengend Snippet: Figure 2. Addition of hydroxyapatite binding (HAB) tag to 30Kc19𝛼-RUNX2 and its effect on cytotoxicity and cell-penetration of 30Kc19𝛼-RUNX2. A) Plasmid construction of 30Kc19𝛼-RUNX2 and HAB-30Kc19𝛼-RUNX2 (left) and chemical structure of HAB (right). B) Coomassie blue staining and western blot analysis of purified recombinant proteins. Anti-RUNX2 antibody was used as a primary antibody. M, marker. C,D) Cytotoxicity assay data showing low cytotoxicity of recombinant proteins. CELLOMAX cell viability assay data taken 72 h after treating cells with various recombinant protein concentrations (C) and Live/Dead images taken 72 h after treating cells with 1 μm of recombinant proteins (D). Quantification of the percentage of live cells is also shown in the bar graph. The percentage of dead cells was presented as fold changes relative to the control group. n.s., non-significant. Scale bar: 100 μm. E) Confocal images of recombinant protein-treated hMSCs showing the cell-penetration ability of recombinant proteins. hMSCs were treated with 1 μm of the proteins for 1 h. The recombinant proteins were labelled with Alexa Fluro 488, and the nucleus with DAPI. White arrows indicate recombinant proteins located in the nucleus. Bottom images show the magnified area of the white box. Scale bar: 100 μm (up) and 50 μm (down).

Article Snippet: Anti-RUNX2 antibody (Santa Cruz Biotechnology, USA) was used as a primary antibody, and goat anti-mouse IgG-HRP antibody (AbFrontier, Korea) was used as a secondary antibody.

Techniques: Binding Assay, Plasmid Preparation, Staining, Western Blot, Recombinant, Marker, Cytotoxicity Assay, Viability Assay, Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Bone-Targeted Delivery of Cell-Penetrating-RUNX2 Fusion Protein in Osteoporosis Model.

doi: 10.1002/advs.202301570

Figure Lengend Snippet: Figure 3. Binding affinity of HAB-30Kc19𝛼-RUNX2 to osteoblast mineral deposits and bone tissue section. A) Confirmation of fluorescent dye (OG488) conjugation to recombinant proteins by measuring the fluorescence intensity of unlabel1ed and label1ed recombinant proteins. B) Comparison of binding affinities of 30Kc19𝛼-RUNX2 and HAB-30Kc19𝛼-RUNX2 to osteoblast mineral deposits. hMSCs were cultured with MSCGM or OM for 3 weeks. Then, cells were fixed with 4% PFA and treated with 1 μm of OG488-label1ed recombinant proteins. Scale bar: 500 μm. C) Comparison of binding affinities of recombinant proteins to a bone tissue section. Mouse tissue sections were treated with 1 μm of OG488-label1ed recombinant proteins. Bright-field and green, fluorescent images were merged using ImageJ software. Scale bar: 500 μm.

Article Snippet: Anti-RUNX2 antibody (Santa Cruz Biotechnology, USA) was used as a primary antibody, and goat anti-mouse IgG-HRP antibody (AbFrontier, Korea) was used as a secondary antibody.

Techniques: Binding Assay, Conjugation Assay, Recombinant, Comparison, Cell Culture, Software

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Bone-Targeted Delivery of Cell-Penetrating-RUNX2 Fusion Protein in Osteoporosis Model.

doi: 10.1002/advs.202301570

Figure Lengend Snippet: Figure 4. In vitro osteogenic differentiation of HAB-30Kc19𝛼-RUNX2-treated hMSCs. A–C) ALP staining and ARS staining data showing ALP expression and calcium deposition in hMSCs. hMSCs were treated with 200 nm of 30Kc19𝛼-RUNX2 or HAB-30Kc19𝛼-RUNX2. ALP staining was performed on hMSCs cultured for (7 and 14) days in OM (A). ARS staining was performed on hMSCs cultured for 21 days in OM (B). ARS quantification is shown in bar graph (C). Scale bar: 500 μm. ***p < 0.001, n.s., non-significant. D) Assessment of osteogenic marker gene expression profile (RUNX2, SPP1, and BGLAP) via RT-qPCR. hMSCs cultured for 7, 14, 21 days in OM were examined. mRNA expression levels of osteogenic marker genes were normalized to GAPDH. Then, the normalized values were expressed as relative fold induction (RFI) over the control group on day 7. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Anti-RUNX2 antibody (Santa Cruz Biotechnology, USA) was used as a primary antibody, and goat anti-mouse IgG-HRP antibody (AbFrontier, Korea) was used as a secondary antibody.

Techniques: In Vitro, Staining, Expressing, Cell Culture, Marker, Gene Expression, Quantitative RT-PCR, Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Bone-Targeted Delivery of Cell-Penetrating-RUNX2 Fusion Protein in Osteoporosis Model.

doi: 10.1002/advs.202301570

Figure Lengend Snippet: Figure 5. Biodistribution of HAB-30Kc19𝛼-RUNX2. IVIS in vivo imaging was taken to track recombinant proteins within the body. The bone-specific delivery of HAB-30Kc19𝛼-RUNX2 and the localization of 30Kc19𝛼-RUNX2 were assessed. Balb/c-nu mice were administered with Atto550-label1ed recombinant proteins (50 μg, 0.25 μg μL−1) in PBS intravenously (n = 3). After 2 h, major organs were collected for fluorescence imaging (554/576 nm Excitation/Emission). For accurate comparison between different groups, the minimum and maximum values of all groups were unified (6.59e6/1.39e7 Minimum/Maximum). The average radiant efficiency of the region of interest (ROI) was measured after imaging.

Article Snippet: Anti-RUNX2 antibody (Santa Cruz Biotechnology, USA) was used as a primary antibody, and goat anti-mouse IgG-HRP antibody (AbFrontier, Korea) was used as a secondary antibody.

Techniques: In Vivo Imaging, Recombinant, Imaging, Comparison

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Bone-Targeted Delivery of Cell-Penetrating-RUNX2 Fusion Protein in Osteoporosis Model.

doi: 10.1002/advs.202301570

Figure Lengend Snippet: Figure 6. In vivo bone regeneration of HAB-30Kc19𝛼-RUNX2 treatment in postmenopausal osteoporosis model. A,B) Micro-CT analysis for new bone formation after recombinant protein treatments. Four weeks after the osteoporosis model was generated by ovariectomy (OVX) in Balb/c mice, PBS (50 μL), 30Kc19𝛼-RUNX2 (50 μg, 1 μg μL−1) and HAB-30Kc19𝛼-RUNX2 (50 μg, 1 μg μL−1) were injected via tail vein injection four times every 1 week (n = 3). The micro-CT image of the distal femur was reconstructed into a 3D image, showing the alternation in trabecular bone density (A). Bone volume to total volume fraction (BV/TV) was also measured (B). *p < 0.05. C) H&E staining showing the histomorphological change in the femur. Scale bar: 1 mm. D) MTC staining showing collagen and mineralized bone formation. Scale bar: 1 mm.

Article Snippet: Anti-RUNX2 antibody (Santa Cruz Biotechnology, USA) was used as a primary antibody, and goat anti-mouse IgG-HRP antibody (AbFrontier, Korea) was used as a secondary antibody.

Techniques: In Vivo, Micro-CT, Recombinant, Generated, Injection, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Bone-Targeted Delivery of Cell-Penetrating-RUNX2 Fusion Protein in Osteoporosis Model.

doi: 10.1002/advs.202301570

Figure Lengend Snippet: Figure 7. Morphological and cytological changes in the spleen after the treatment of recombinant proteins. A) Splenomegaly observed after the treatment of 30Kc19𝛼-RUNX2 and HAB-30Kc19𝛼-RUNX2. Following 4 weeks of protein injection, spleens were dissected to evaluate protein delivery to the spleen and the immune response induced by external protein injection. Scale bar: 10 mm B) H&E staining showing the cellular and histomorphological change in the spleen.

Article Snippet: Anti-RUNX2 antibody (Santa Cruz Biotechnology, USA) was used as a primary antibody, and goat anti-mouse IgG-HRP antibody (AbFrontier, Korea) was used as a secondary antibody.

Techniques: Recombinant, Injection, Staining