Journal: Bone & Joint Research

Article Title: OX40L in endothelial cells promotes temporomandibular joint subchondral bone angiogenesis and osteoclastogenesis in mice

doi: 10.1302/2046-3758.154.BJR-2025-0249.R1

Figure Lengend Snippet: Abnormal activation of OX40L in endothelial cells can promote angiogenesis and osteoclast induction. Human bone microvascular endothelial cells were pretreated with KY1005 for six hours, followed by treatment with sOX40 for 48 hours. a) Detection of vascular endothelial growth factor (VEGF) gene expression using quantitative polymerase chain reaction (qPCR). b) Detection of VEGF protein expression using western blotting. c) Representative images of endothelial cell scratch experiments. Scale bar: 100 µm. d) Representative images of endothelial cell tubule formation experiments. Scale bar: 100 µm. e) Representative image of tartrate-resistant acid phosphatase (TRAP) staining after endothelial cells were co-cultured with RAW264.7 cells. Scale bar: 50 µm. f) Bone resorption pits were shown by scanning electron microscopy. Scale bar: 500 nm.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (Beyotime) and incubated overnight at 4°C with specific primary antibodies against vascular endothelial growth factor (VEGF) (1:1,000; Cat# 19003-1-AP, Proteintech, China) and glyceraldehyde 3-phosphate dehydrogenase (1:2,000; Cat# 60004-1-Ig, Clone 1E6D9, Proteintech).

Techniques: Activation Assay, Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Staining, Cell Culture, Electron Microscopy

Journal: Bone & Joint Research

Article Title: OX40L in endothelial cells promotes temporomandibular joint subchondral bone angiogenesis and osteoclastogenesis in mice

doi: 10.1302/2046-3758.154.BJR-2025-0249.R1

Figure Lengend Snippet: a) Statistical analysis of VEGF protein expression levels. b) Statistical analysis of mobility in scratch experiments. c) and d) Statistical analysis of lumen number and main branch length in tubule formation experiments. e) to h) Detection of osteoprotegerin (OPG), receptor activator of nuclear factor κB ligand (RANKL), and macrophage colony-stimulating factor (M-CSF) secretion in endothelial cell supernatants by enzyme-linked immunosorbent assay (ELISA). i) Statistical analysis of resorption areas. Data are presented as the mean (SD) (n = 3). Statistical significance was determined by one-way analysis of variance followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: The membrane was blocked with 5% bovine serum albumin (Beyotime) and incubated overnight at 4°C with specific primary antibodies against vascular endothelial growth factor (VEGF) (1:1,000; Cat# 19003-1-AP, Proteintech, China) and glyceraldehyde 3-phosphate dehydrogenase (1:2,000; Cat# 60004-1-Ig, Clone 1E6D9, Proteintech).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Communications Biology

Article Title: OPN3-mediated positive regulation of angiogenesis in HUVECs through VEGFR2 interaction

doi: 10.1038/s42003-025-07958-4

Figure Lengend Snippet: a The volcano plot of differentially expressed genes based on transcriptome sequencing results shows the fold change of gene expression (log2FoldChange) on the horizontal axis, representing the expression fold change between the LV-OPN3-RNAi and LV-control-RNAi. The vertical axis represents the significance level of the gene expression difference between the two groups (-log10pvalue). Red dots indicate upregulated genes, while green dots indicate downregulated genes. b The scatter plot of GO functional enrichment analysis based on transcriptome sequencing results is divided into three categories: biological process, cellular component, and molecular function. A total of 30 significant terms are selected from the GO enrichment analysis results for visualization, with a threshold of padj < 0.05 to define significant enrichment. The horizontal axis represents the ratio of the number of differentially expressed genes annotated to the GO term to the total number of differentially expressed genes. The vertical axis represents the GO terms. The size of the dots corresponds to the number of genes annotated to the GO term, and the color gradient from red to purple reflects the significance of enrichment. c Based on the transcriptome sequencing results, hierarchical clustering was performed on the FPKM values of the genes, and the row (genes) was normalized (Z-score). The resulting heatmap clusters genes with similar expression patterns together. In the heatmap, the colors represent the expression levels of the same gene across different samples, and each square’s color reflects the normalized expression value (ranging from −1.5 to 1.5). Red indicates higher expression, and blue indicates lower expression. d , e RT-qPCR was used to detect the mRNA expression levels of OPN3 and VEGFR2 after OPN3 knockdown and overexpression in HUVECs. The relative mRNA expression levels were calculated using the 2 −ΔΔCt method, with GAPDH serving as the internal control ( n = 3 independent experiments, with each experimental group consisting of 6 dishes, derived from 3 different donors, with each donor providing 2 dishes of cells). Statistical analysis was performed using an unpaired t-test: **p < 0.01, ***p < 0.001, ****p < 0.0001 .

Article Snippet: Protein analysis was performed by Western blotting using primary antibodies against VEGFR2 (55B11, Rabbit, 1:1000, Cell Signaling Technology, USA) and OPN3 (A15803, Rabbit, 1:500, ABclonal, China).

Techniques: Sequencing, Gene Expression, Expressing, Control, Functional Assay, Quantitative RT-PCR, Knockdown, Over Expression, Derivative Assay

Journal: Communications Biology

Article Title: OPN3-mediated positive regulation of angiogenesis in HUVECs through VEGFR2 interaction

doi: 10.1038/s42003-025-07958-4

Figure Lengend Snippet: a , b Western blot analysis was used to detect the protein expression of OPN3 and VEGFR2 after lentiviral knockdown and overexpression of OPN3, using β-tubulin as a loading control for normalization in the WB analysis. Relative protein levels were quantified using ImageJ software ( n = 3 independent experiments, with each experimental group consisting of 6 dishes, derived from 3 different donors, with each donor providing 2 dishes of cells). Statistical analysis was performed using an unpaired t-test: ns (not significant), *p < 0.05, ***p < 0.001 . c SiRNA was used to silence VEGFR2 expression in HUVECs. The experiment was divided into a knockdown group (RNAi-VEGFR2) and a control group (RNAi-control). RT-qPCR was used to detect the mRNA expression levels of VEGFR2 under different siRNA concentrations (30 nM, 50 nM, 70 nM). The relative mRNA expression levels were calculated using the 2 −ΔΔCt method, with GAPDH serving as the internal control ( n = 3 independent experiments, with each experimental group consisting of 6 dishes, derived from 3 different donors, with each donor providing 2 dishes of cells). Statistical analysis was performed using an unpaired t-test: ns (not significant), **p < 0.01, ****p < 0.0001 . d Western blot analysis was used to detect VEGFR2 protein expression under 50 nM siRNA treatment, using β-tubulin as a loading control for normalization in the WB analysis. Relative protein levels were quantified using ImageJ software ( n = 3 independent experiments, with each experimental group consisting of 6 dishes, derived from 3 different donors, with each donor providing 2 dishes of cells). Statistical analysis was performed using an unpaired t-test: *p < 0.05 . e Western blot analysis was used to detect OPN3 and VEGFR2 protein expression levels in different cell groups with overexpression of OPN3 or simultaneous overexpression of OPN3 and knockdown of VEGFR2, using β-tubulin as a loading control for normalization in the WB analysis. Relative protein levels were quantified using ImageJ software ( n = 3 independent experiments, with each experimental group consisting of 6 dishes, derived from 3 different donors, with each donor providing 2 dishes of cells). Statistical analysis was performed using an unpaired t-test: ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001 . f , h Cells from different experimental groups were seeded on Matrigel for 10 h to record and quantify tube formation. The number and length of branches were analyzed using ImageJ software to determine the extent of tube formation. f , g Spheroid sprouting experiments were conducted with cells from different experimental groups. The sprouts were recorded and quantified using optical microscopy, and the number and length of sprouts were analyzed using ImageJ software to evaluate the sprouting ability of HUVECs ( n = 9 dishes of cultured HUVECs from 3 different donors, with each donor replicated three times). Statistical analysis was performed using an unpaired t-test: ns (not significant), ****p < 0.0001 . The scale bar represents 100 μm. Data are presented as mean ± SEM.

Article Snippet: Protein analysis was performed by Western blotting using primary antibodies against VEGFR2 (55B11, Rabbit, 1:1000, Cell Signaling Technology, USA) and OPN3 (A15803, Rabbit, 1:500, ABclonal, China).

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Control, Software, Derivative Assay, Quantitative RT-PCR, Microscopy, Cell Culture

Journal: Communications Biology

Article Title: OPN3-mediated positive regulation of angiogenesis in HUVECs through VEGFR2 interaction

doi: 10.1038/s42003-025-07958-4

Figure Lengend Snippet: a , b Western blot analysis was used to detect the protein levels of OPN3 and VEGFR2 in different cell groups with lentiviral knockdown of OPN3 or lentiviral knockdown of OPN3 combined with plasmid overexpression of OPN3 (GFP-OPN3), using β-tubulin as a loading control for normalization in the WB analysis. Relative protein levels were quantified using ImageJ software ( n = 3 independent experiments, with each experimental group consisting of 6 dishes, derived from 3 different donors, with each donor providing 2 dishes of cells). Statistical analysis was performed using an unpaired t-test: ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001 . c , d Western blot analysis was used to detect the protein levels of OPN3 and VEGFR2 in different cell groups with lentiviral overexpression of OPN3 or overexpression of OPN3 combined with siRNA-mediated knockdown of OPN3 (RNAi-OPN3), using β-tubulin as a loading control for normalization in the WB analysis. Relative protein levels were quantified using ImageJ software ( n = 3 independent experiments, with each experimental group consisting of 6 dishes, derived from 3 different donors, with each donor providing 2 dishes of cells). Statistical analysis was performed using an unpaired t-test: *p < 0.05, **p < 0.01 . e HUVECs were stimulated with VEGF (25 ng/mL), and cells were collected at different time points (5 min, 15 min, 30 min) to detect the interaction between OPN3 and VEGFR2 by Co-IP. Anti-OPN3 or anti-IgG (negative control) antibodies were used for immunoprecipitation (IP), followed by western blot analysis to detect OPN3 and VEGFR2 protein levels. Relative protein levels were quantified using ImageJ software. f Bar graph represents the averaged fold change of VEGFR2/OPN3 ratio over the basal ratio ( n = 3 independent experiments, with each experimental group consisting of 12 dishes, derived from 6 different donors, with each donor providing 2 dishes of cells). Statistical analysis was performed using an unpaired t-test: *p < 0.05, **p < 0.01 . g , h HUVECs were stimulated or not stimulated with VEGF for 15 min, followed by co-staining of OPN3 and VEGFR2. Images were captured using confocal microscopy. OPN3 was labeled in green, VEGFR2 in red, and DAPI in blue. The scale bar represents 25 μm. Yellow fluorescence (indicated by white arrows) in the merged images indicates their colocalization, which was analyzed by comparing the fluorescence intensity of each protein along the white line in the magnified images of the white box. A bar graph shows the percentage of colocalization ( n = 6 dishes of cultured HUVECs from 3 different donors, with each donor replicated twice). Statistical analysis was performed using an unpaired t-test: ***p < 0.001 .

Article Snippet: Protein analysis was performed by Western blotting using primary antibodies against VEGFR2 (55B11, Rabbit, 1:1000, Cell Signaling Technology, USA) and OPN3 (A15803, Rabbit, 1:500, ABclonal, China).

Techniques: Western Blot, Knockdown, Plasmid Preparation, Over Expression, Control, Software, Derivative Assay, Co-Immunoprecipitation Assay, Negative Control, Immunoprecipitation, Staining, Confocal Microscopy, Labeling, Fluorescence, Cell Culture

Journal: Communications Biology

Article Title: OPN3-mediated positive regulation of angiogenesis in HUVECs through VEGFR2 interaction

doi: 10.1038/s42003-025-07958-4

Figure Lengend Snippet: a Western blot analysis of OPN3 and VEGFR2 protein expression in fli1 :EGFP zebrafish embryos after OPN3 knockdown using MO. β-tubulin was used as a loading control for normalization in the WB analysis. Relative protein levels were quantified using ImageJ ( n = 9 batches of injected zebrafish embryos, each batch containing 30 embryos). Statistical analysis was performed using an unpaired t-test: ****p < 0.0001 . b Western blot analysis of OPN3 and VEGFR2 protein expression in WT and OPN3 -/- at 48 hpf. β-tubulin was used as a loading control for normalization in the WB analysis. Relative protein levels were quantified using ImageJ ( n = 9 batches of injected zebrafish embryos, each batch containing 30 embryos). Statistical analysis was performed using an unpaired t-test: ****p < 0.0001 . c Western blot analysis of OPN3 and VEGFR2 protein expression in kdrl :mCherry zebrafish embryos after OPN3 knockdown using MO, and after co-injection of OPN3 MO with OPN3 mRNA. β-tubulin was used as a loading control for normalization in the WB analysis. Relative protein levels were quantified using ImageJ ( n = 9 batches of injected zebrafish embryos, each batch containing 30 embryos). Statistical analysis was performed using an unpaired t-test: **p < 0.01, ***p < 0.001, ****p < 0.0001 . d Inject kdrl :mCherry transgenic zebrafish embryos with Control MO, OPN3 MO, or OPN3 MO and OPN3 mRNA, and image the treated embryos from each group at 48 hpf using a confocal microscope. The fluorescence of kdrl is absent and discontinuous in many places (*). e Use ImageJ to analyze the fluorescence intensity in the images from each group ( n = 15, each individual data point represents the trunk or brain region of a single zebrafish embryo). Statistical analysis was performed using an unpaired t-test: ****p < 0.0001 . The scale bar represents 100 μm. f Inject fli1 :EGFP transgenic zebrafish embryos with Control MO, OPN3 MO, or OPN3 MO and SC79, and image the treated embryos from each group at 48 hpf using a confocal microscope. DLAVs and ISVs were absent and discontinuous in many places (**). g Use ImageJ to analyze the normalized vessel length and the number of junctions in the images from each group ( n = 15, each individual data point represents the trunk or brain region of a single zebrafish embryo). Statistical analysis was performed using an unpaired t-test: ****p < 0.0001 . The scale bar represents 100 μm. h Western blot analysis of p-AKT protein expression in fli1 :EGFP zebrafish embryos after OPN3 knockdown using MO, and after co-injection of OPN3 MO with SC79. β-tubulin was used as a loading control for normalization in the WB analysis. i Relative protein levels were quantified using ImageJ ( n = 9 batches of injected zebrafish embryos, each batch containing 30 embryos). Statistical analysis was performed using an unpaired t-test: ***p < 0.001, ****p < 0.0001 . Data are presented as mean ± SEM.

Article Snippet: Protein analysis was performed by Western blotting using primary antibodies against VEGFR2 (55B11, Rabbit, 1:1000, Cell Signaling Technology, USA) and OPN3 (A15803, Rabbit, 1:500, ABclonal, China).

Techniques: Western Blot, Expressing, Knockdown, Control, Injection, Transgenic Assay, Microscopy, Fluorescence