Journal: bioRxiv

Article Title: Functional impact of pathogenic Runt domain mutations in Runx2 in vivo : Insights into the skeletal and dental anomalies of cleidocranial dysplasia

doi: 10.1101/2025.06.18.660258

Figure Lengend Snippet: Generation of Runx2 mutant mice using Platinum TALENs. a Schematic representation of the mouse Runx2 gene with eight exons (labeled E1-E8 below), distal (P1) and proximal (P2) promoters for Type II (MASNSL) and Type I (MRIVD) isoforms, and target sites of Platinum TALENs ( mRunx2-TALEN-L and -R ). A 10 kb scale bar is shown. The SmaI site (red box) includes G695 (underlined). The ssODN contains target sites (green), a T>G silent mutation at 690 creating an ApaI site (blue box), and a G>A mutation at 695 disrupting the SmaI site. b Domain structure of the mRunx2 Type II protein (528 aa) and its corresponding exons. The protein comprises a QA repeat domain, RHD, NLS, PST-rich domain, NMTS, and a C-terminal VWRPY motif. Amino acid positions are indicated above, and the corresponding exons (E1-E8) are shown below. c Nucleotide sequences are shown for the wild-type ( Wt , 670-725), missense mutant ( m , c.695G>A; p.R232Q, 670-725), and two-base deletion ( Δ2 , c.697_698delGA; p.E233TfsTer9, 670-723) alleles. Wt is on top, m in the middle, and Δ2 at the bottom. d Representative electropherograms of the targeted Runx2 region in Runx2 +/+ , Runx2 m/+ , and Runx2 Δ2/+ mice. A black arrow marks T at 690 in Runx2 +/+ and Runx2 m/+ mice. A blue arrow indicates overlapping T and A peaks at 690 in Runx2 m/+ mice, and a red arrow shows overlapping A and G peaks at position 695 in Runx2 m/+ mice.

Article Snippet: After blocking with 2% skim milk/PBS, sections were incubated with rabbit anti-Runx2 monoclonal antibody (1:1600, Cell Signaling Technology) or rabbit anti-Collagen X monoclonal antibody (1:500, Abcam, Cambridge, UK) overnight, washed with PBS, and incubated with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary antibody conjugated with Alexa Fluor Plus 594 (1:500, Thermo Fisher Scientific).

Techniques: Mutagenesis, TALENs, Labeling

Journal: bioRxiv

Article Title: Functional impact of pathogenic Runt domain mutations in Runx2 in vivo : Insights into the skeletal and dental anomalies of cleidocranial dysplasia

doi: 10.1101/2025.06.18.660258

Figure Lengend Snippet: Skeletal phenotypes of Runx2 mutant mice. a-j Skeletal preparations of Runx2 +/+ ( a, f, g ), Runx2 m/+ ( b, h, i ), Runx2 Δ2/+ ( c ), Runx2 m/m ( d, j ), and Runx2 Δ2/Δ2 ( e ) mice at E18.5. Lateral views of embryos ( a-e ), clavicles ( g, i ) and dorsal views of the calvaria ( f, h, j ) are presented. A white arrow indicates weak calcification. Scale bars, 2 mm.

Article Snippet: After blocking with 2% skim milk/PBS, sections were incubated with rabbit anti-Runx2 monoclonal antibody (1:1600, Cell Signaling Technology) or rabbit anti-Collagen X monoclonal antibody (1:500, Abcam, Cambridge, UK) overnight, washed with PBS, and incubated with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary antibody conjugated with Alexa Fluor Plus 594 (1:500, Thermo Fisher Scientific).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Functional impact of pathogenic Runt domain mutations in Runx2 in vivo : Insights into the skeletal and dental anomalies of cleidocranial dysplasia

doi: 10.1101/2025.06.18.660258

Figure Lengend Snippet: Histology and in situ hybridization of forearms in wild-type and Runx2 mutant mice. TB, ALP, and AR staining of forearms prepared from Runx2 +/+ , Runx2 m/+ , Runx2 m/m and Runx2 Δ2/Δ2 embryos at E18.5. d-j Expression of Col2a1 ( d ), Acan ( e ), Ihh ( f ), Col10a1 ( g ), Runx2 ( h ), Spp1 ( i ), and Col1a1 ( j ) in the forearm of Runx2 +/+ , Runx2 m/+ , or Runx2 m/m embryos at E18.5. Blue arrowheads in ( f ), ( g ), and ( i ) mark prehypertrophic/hypertrophic chondrocytes. A red arrowhead in ( h ) indicates the perichondrium. Scale bars, 500 µm.

Article Snippet: After blocking with 2% skim milk/PBS, sections were incubated with rabbit anti-Runx2 monoclonal antibody (1:1600, Cell Signaling Technology) or rabbit anti-Collagen X monoclonal antibody (1:500, Abcam, Cambridge, UK) overnight, washed with PBS, and incubated with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary antibody conjugated with Alexa Fluor Plus 594 (1:500, Thermo Fisher Scientific).

Techniques: In Situ Hybridization, Mutagenesis, Staining, Expressing

Journal: bioRxiv

Article Title: Functional impact of pathogenic Runt domain mutations in Runx2 in vivo : Insights into the skeletal and dental anomalies of cleidocranial dysplasia

doi: 10.1101/2025.06.18.660258

Figure Lengend Snippet: Expression and transcriptional activity of wild-type and mutant Runx2. a Expression of wild-type and missense mutant Runx2 in HEK293T cells. Cells were transfected with an empty vector as the control, wild-type Runx2, or the Runx2 RQ mutant construct, all using the pcDNA3 vector. b Expression of Runx2 in forelimb extracts from Runx2 +/+ , Runx2 m/+ and Runx2 m/m mice at E18.5. β-Actin was used as a loading control in both a and b . c Dual luciferase assay in HEK293T cells co-transfected with the p6OSE2-luc reporter vector and either empty vector, Runx2 WT, Runx2 RQ, RW, or RX mutant constructs using the pcDNA3 vector . pGL4.74[hRluc/TK] was also co-transfected for normalization. Values for each of five wells were normalized to pGL4.74[hRluc/TK] and are shown as fold induction relative to the empty vector. Data present one of ≧3 independent experiments (mean ± SD, n=3). **** P < 0.0001 vs. Runx2 WT .

Article Snippet: After blocking with 2% skim milk/PBS, sections were incubated with rabbit anti-Runx2 monoclonal antibody (1:1600, Cell Signaling Technology) or rabbit anti-Collagen X monoclonal antibody (1:500, Abcam, Cambridge, UK) overnight, washed with PBS, and incubated with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary antibody conjugated with Alexa Fluor Plus 594 (1:500, Thermo Fisher Scientific).

Techniques: Expressing, Activity Assay, Mutagenesis, Transfection, Plasmid Preparation, Control, Construct, Luciferase

Journal: bioRxiv

Article Title: Functional impact of pathogenic Runt domain mutations in Runx2 in vivo : Insights into the skeletal and dental anomalies of cleidocranial dysplasia

doi: 10.1101/2025.06.18.660258

Figure Lengend Snippet: Altered expression and subcellular localization of mutant Runx2 in chondrocytes. a-c Immunostaining of Col10 ( a, c ) and Runx2 ( b, d ) proteins in the ulna of Runx2 +/+ and Runx2 m/m embryos at E18.5. Nuclei are counterstained with DAPI. Yellow boxes in ( a ) and ( c ) indicate the Col10 + prehypertrophic/hypertrophic cartilage zone. The corresponding regions to them are shown in the semi-serial sections in ( b ) and ( d ). The number of cells in the yellow boxes of ( a ) and ( c ) indicating Col10 + region differs between Runx2 +/+ and Runx2 m/m . e-h Confocal images of wild-type and mutant Runx2 (red) and nuclei (blue) in prehypertrophic/hypertrophic chondrocytes. Representative localization patterns of wild-type ( e, f ) and mutant ( g, h ) Runx2 are highlighted by rectangles and shown in insets. i Nuclear localization rate of Runx2 in chondrocytes of the ulna in Runx2 +/+ and Runx2 m/m embryos. Runx2 signal intensities in the whole-cell (dashed regions in f and h) and those in nucleus were quantified using imageJ. Data are means ± SD ( Runx2 +/+ , n = 88 cells; Runx2 m/m , n = 45 cells). ****P < 0.0001 (Mann-Whitney U -test). Scale bars, 200 µm (a-d), 50 µm (e-h).

Article Snippet: After blocking with 2% skim milk/PBS, sections were incubated with rabbit anti-Runx2 monoclonal antibody (1:1600, Cell Signaling Technology) or rabbit anti-Collagen X monoclonal antibody (1:500, Abcam, Cambridge, UK) overnight, washed with PBS, and incubated with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary antibody conjugated with Alexa Fluor Plus 594 (1:500, Thermo Fisher Scientific).

Techniques: Expressing, Mutagenesis, Immunostaining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Functional impact of pathogenic Runt domain mutations in Runx2 in vivo : Insights into the skeletal and dental anomalies of cleidocranial dysplasia

doi: 10.1101/2025.06.18.660258

Figure Lengend Snippet: Postnatal skeletal development in Runx2 +/+ and Runx2 m/+ mice. a Gross appearance of Runx2 +/+ and Runx2 m/+ mice at P30. b Body weight of Runx2 +/+ and Runx2 m/+ mice at 16 weeks (n=3). *P < 0.05 vs. Runx2 +/+ (unpaired Student’s t -test). c µCT- based 3D reconstruction images of the axial and appendicular skeleton in the upper body of 3- month-old Runx2 +/+ and Runx2 m/+ mice. d Skeletal preparations of the pectoral girdle and upper limb in Runx2 +/+ and Runx2 m/+ mice at 6 months. e µCT-based 3D reconstruction images of the calvaria in Runx2 +/+ and Runx2 m/+ mice at 1, 2, and 3 months. f Skeletal preparations of the skull from Runx2 +/+ and Runx2 m/+ mice at 6 months. Abbreviations: sc, scapula; cl, clavicle; hu, humerus; f, frontal bone; p, parietal bone; ip, interparietal bone; o, occipital bone. Scale bars, 5 mm.

Article Snippet: After blocking with 2% skim milk/PBS, sections were incubated with rabbit anti-Runx2 monoclonal antibody (1:1600, Cell Signaling Technology) or rabbit anti-Collagen X monoclonal antibody (1:500, Abcam, Cambridge, UK) overnight, washed with PBS, and incubated with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary antibody conjugated with Alexa Fluor Plus 594 (1:500, Thermo Fisher Scientific).

Techniques:

Journal: bioRxiv

Article Title: Functional impact of pathogenic Runt domain mutations in Runx2 in vivo : Insights into the skeletal and dental anomalies of cleidocranial dysplasia

doi: 10.1101/2025.06.18.660258

Figure Lengend Snippet: Dental phenotypes of postnatal Runx2 missense mice. a-d 3D µCT images of the maxilla and mandible in Runx2 +/+ and Runx2 m/+ mice at 3 months. Panels ( a ) and ( c ) show whole jaw views. Panels ( b ) and ( d ) show slice images with overlayed 3D reconstructions of the maxillary first molar. Insets display enlarged views of the molars. A red arrow in ( d ) indicates an additional root-like structure. Scale bars, 2 mm. e-l 3D µCT images of the right maxillary first molars in Runx2 m/+ mice at 2, 3, 4, and 8 weeks. Axial ( e, g, i, k ) and corresponding sagittal ( f, h, j, l ) views from the same individuals are shown. Orange lines in axial panels indicate the sagittal section planes. Directional arrows indicate the palatal (P), buccal (B), apical (A), occlusal (O), distal (D) and mesial (M) directions. Images are from the same individuals (n=3). m Schematic illustration of root furcation in Runx2 +/+ and Runx2 m/+ mice. Horizontal (top) and sagittal (bottom) views are shown, with orange lines in ( e - k ) indicating the sagittal section planes. Tissue types are presented by pattern-filled squares. The green panel shows the fusion of three epithelial processes (two buccal and one palatal) at P9, P11, and P12. The blue and pink panels depict the progression of root furcation at 2, 3, and 4 weeks in Runx2 +/+ and Runx2 m/+ mice, respectively.

Article Snippet: After blocking with 2% skim milk/PBS, sections were incubated with rabbit anti-Runx2 monoclonal antibody (1:1600, Cell Signaling Technology) or rabbit anti-Collagen X monoclonal antibody (1:500, Abcam, Cambridge, UK) overnight, washed with PBS, and incubated with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary antibody conjugated with Alexa Fluor Plus 594 (1:500, Thermo Fisher Scientific).

Techniques:

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Pharmacological inhibition of PPARγ increases osteoblastogenesis and bone mass in male C57BL/6 mice.

doi: 10.1002/jbmr.1782

Figure Lengend Snippet: Fig. 3. Treatment with PPARg inhibitors in vivo results in increased osteoblastogenesis in bone marrow stromal cells (BMSC) cultured ex vivo. (A, B) Formation of colony-forming units–osteoblasts (CFU-OB) in ex vivo cultures of bone marrow cells from 9-month-old C57BL/6 male mice treated for 6 weeks with either BADGE, BADGE þ 1,25(OH)3, or vehicle alone. The number of CFU-OB per femur was significantly higher after 2 weeks of differentiation in both BADGE- and BADGE þ 1,25(OH)3- treated mice as compared with mice treated with vehicle alone. p < 0.01. (C, D) Treatment with BADGE alone or in combination with 1,25(OH)3 induced significant levels of protein expression for the osteogenic transcription factors osteocalcin (OCN) and Runx2. In contrast, levels of expression of osteopontin (OPN) were decreased after treatment with BADGE and BADGE þ 1,25(OH)3. Data from scanning densitometric analyses is expressed as the ratio of the protein of interest in the BADGE and BADGE þ 1,25(OH)3 mice using the values of vehicle- treated mice as controls representing the mean SD of triplicate determinations. p < 0.01. (E) Runx2 DNA-binding activity was determined using ELISA- based Runx2 activation kit and quantified by colorimetry. The figure shows the levels of activity after treatment with either BADGE and BADGE þ 1,25(OH)3 or vehicle alone. Treatment with BADGE alone or in combination with 1,25(OH)3 significantly increased the activity of the Runx2 complex in the nuclei. Values are mean SEM of protein extracts obtained from marrow fat of 8 mice per group. p < 0.01.

Article Snippet: After blocking with PBS containing 0.1% Tween 20 and 10% nonfat dry milk, membranes were incubated overnight at 48C using mouse monoclonal antibodies directed against Runx2, OCN, osteopontin (OPN), PPARg, CCAAT/enhancer-binding protein alpha (CEBPa), and sterol regulatory elementbinding protein-1 (SREBP-1) (1:1000, Santa Cruz Biotechnology).

Techniques: In Vivo, Cell Culture, Ex Vivo, Expressing, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Colorimetric Assay

Journal: Bioactive Materials

Article Title: A multimodal ROS logic-gated therapeutic platform disrupts the vicious cycle of senescence to promote aged bone defect repair

doi: 10.1016/j.bioactmat.2026.02.002

Figure Lengend Snippet: Therapy platform enhances osteogenic potential of senescent BMSCs in vitro . (A) Representative ALP staining images at day 7 (scale bars: top-10 mm, and bot-200 μm). (B) Quantitative analysis of ALP activity ( n = 3). (C) Representative ARS staining images at day 21 (scale bars: top-5 mm and bot-200 μm). (D) Quantification of mineralized matrix formation via ARS staining ( n = 3). (E-H) qRT-PCR analysis of osteogenic markers ( Alp , Osx , Runx2 , and Opn ) in BMSCs ( n = 3). (I) Representative immunofluorescence images of RUNX2 staining (scale bars: 50 μm). (J) Quantitative analysis of RUNX2 fluorescence intensity ( n = 3). (K) Representative immunofluorescence images of OPN staining (scale bars: 50 μm). (L) Quantitative analysis of OPN fluorescence intensity ( n = 3). Data are presented as mean ± SD; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Protein expression of RUNX2 (Proteintech, 20700-1-AP; 1:500) and OPN (Abcam, ab63856; 1:500) were detected by immunofluorescence staining on day 14, and fluorescence intensities were quantified using ImageJ software (Media Cybernetics).

Techniques: In Vitro, Staining, Activity Assay, Quantitative RT-PCR, Immunofluorescence, Fluorescence

Journal: Bioactive Materials

Article Title: A multimodal ROS logic-gated therapeutic platform disrupts the vicious cycle of senescence to promote aged bone defect repair

doi: 10.1016/j.bioactmat.2026.02.002

Figure Lengend Snippet: In vivo evaluation of the therapeutic platform for aged bone defect repair. (A) Representative 3D-reconstructed micro-CT images of calvarial bone defects in different groups (scale bars: 2 mm). (B-C) Quantification of bone volume fraction (BV/TV) and bone mineral density (BMD) based on micro-CT analysis ( n = 5). (D-E) Representative histological images of H&E staining (D) and Masson's trichrome staining (E). Low-magnification images (scale bars: 500 μm) show the overall defect region, while high-magnification images (scale bars: 100 μm) highlight the new bone (blue box) and defect-host bone interface (red box). Green arrows indicate defect boundaries; FT: fiber tissue; NB: new formed bone; HB: host bone. (F-G) Immunofluorescence images and quantification of RUNX2 expression in defect areas (scale bars: 50 μm, n = 3). (H-I) Immunofluorescence images and quantification of OCN expression (scale bars: 50 μm, n = 3). (J) Schematic illustration of the programmed ROS bidirectional modulation strategy for infection control and repair of aged bone defects, depicting acousto-optic dynamic ROS generation for antimicrobial effects followed by antioxidant modulation to rejuvenate senescent BMSCs and promote aged bone regeneration. Data are expressed as mean ± SD; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Protein expression of RUNX2 (Proteintech, 20700-1-AP; 1:500) and OPN (Abcam, ab63856; 1:500) were detected by immunofluorescence staining on day 14, and fluorescence intensities were quantified using ImageJ software (Media Cybernetics).

Techniques: In Vivo, Micro-CT, Staining, Immunofluorescence, Expressing, Infection, Control