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: Discover Oncology

Article Title: Anticancer effect of the antirheumatic drug leflunomide on oral squamous cell carcinoma by the inhibition of tumor angiogenesis

doi: 10.1007/s12672-025-01763-5

Figure Lengend Snippet: Immunohistochemistry study of tumor specimens of syngeneic mice. a Representative microscopic images of the specimen of syngeneic mice. HE staining and IHC staining for DHODH and the tumor vascular endothelial markers integrin αv, VEGFR2, and CD31 of the tumors from the control (DMSO vehicle control) and LEF-treated (LEF-treated group) groups. Ten fields of view were taken for each specimen, and images of representative fields of view (sample number 4, 5 as control and 8, 9 as test in Fig. ) are shown on the left at low magnification and right at high magnification of the areas highlighted in white rectangle. Black bars in all panels indicated 500 µm. ( b ) Quantitative analysis of IHC positivity rates was conducted by calculating the ratio of the stained area of each marker, DHODH and vascular endothelial cells, to the horizontal projected area of the sections. This analysis was performed using BZ-X Analyzer software (BZ-H4A, Keyence Corporation, Osaka, Japan), with five sections randomly selected for each marker. Statistical analysis was performed using Student’s t-test. Values are expressed as means ± SEM of five sections (* p < 0.05)

Article Snippet: Immunostaining was performed using primary antibodies against vascular endothelial growth factor receptor 2 (VEGFR2) (rabbit mAb, 55B11; at 1: 100, Cell Signaling Technology, Danvers, MA, US), integrin alpha V rabbit mAb (ab179475; at 1: 500), CD31 mouse mAb (ab182981; at 1: 2000, Abcam Inc., Cambridge, MA, USA), DHODH rabbit pAb (14877-1-AP; at 1: 300, Proteintech, Rosemont, IL, USA).

Techniques: Immunohistochemistry, Staining, Control, Marker, Software

Journal: Frontiers in Immunology

Article Title: miR-210-3p protects against osteoarthritis through inhibiting subchondral angiogenesis by targeting the expression of TGFBR1 and ID4

doi: 10.3389/fimmu.2022.982278

Figure Lengend Snippet: miR-210-3p directly targets TGFBR1 and ID4. (A–C) ID4, TGFBR1 and CXCL1 mRNA expression levels quantified by RT–qPCR. (D–F) Representative bands showing the levels of the VEGF, ID4, CCL2, TGFBR1, Smad2, pSmad2, Smad3, pSmad3 and GAPDH proteins (D) . The relative VEGF, ID4, CCL2 and TGFBR1 protein levels were normalized to the GAPDH level. The relative pSmad2 and pSmad3 protein levels were normalized to those of Smad2 and Smad3, respectively (E, F) . (G) Predicted binding sites for miR-210-3p in the ID4 and TGFBR1 sequences. (H, I) A dual-luciferase reporter assay showed that luciferase activity was significantly inhibited in groups cotransfected with ID4 or TGFBR1 WT vectors and miR-210-3p mimic. ** P < 0.01. * P < 0.05. WT, wild-type. Mu, mutant. ns, not significance. All data are presented as the means ± standard deviations. Statistical significance was determined using unpaired, 2-tailed Student’s t test.

Article Snippet: The membranes were then incubated with primary antibodies against vascular endothelial growth factor (VEGF; Servicebio, Wuhan, China), TGFBR1 (Abcam, Cambridge, UK), ID4 (Santa Cruz, Dallas, Texas, USA), C-C motif chemokine ligand 2 (CCL2; Huabio, Hangzhou, China), Smad2 (Santa Cruz, Dallas, Texas, USA), pSmad2 (CST, Danvers, Massachusetts, USA), Smad3 (CST, Danvers, Massachusetts, USA), pSmad3 (CST, Danvers, Massachusetts, USA) and GAPDH (CST, Danvers, Massachusetts, USA) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Reporter Assay, Activity Assay, Mutagenesis