Journal: Experimental and Therapeutic Medicine

Article Title: Negative pressure wound therapy enhances bone regeneration compared with conventional therapy in a rabbit radius gap-healing model

doi: 10.3892/etm.2021.9905

Figure Lengend Snippet: VEGF, BMP-2 and OPN protein expression. (A) Representative western blot image and quantification of (B) VEGF, (C) BMP-2 and (D) OPN protein expression at each time point examined in the NPWT and control groups. * P<0.05 and ** P<0.01. VEGF, vascular endothelial growth factor; BMP-2, bone morphogenetic protein 2; OPN, osteopontin; NPWT, negative pressure wound therapy; 2W, week 2; 4W, week 4.

Article Snippet: The membranes were then incubated overnight at 4˚C with TBS-Tween-20 supplemented with one of the following primary antibodies: Mouse anti-β-actin (1:1,000; Boster Biological Technology; cat. no. P60709), mouse anti-VEGF (1:200; Santa Cruz Biotechnology, Inc. cat. no. sc-7269), mouse anti-BMP-2 (1:400; Santa Cruz Biotechnology, Inc. cat. no. sc-137087) or mouse anti-OPN (1:400; Abcam; cat. no. ab228748).

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Pro-Calcific Environment Impairs Ischaemia-Driven Angiogenesis

doi: 10.3390/ijms23063363

Figure Lengend Snippet: Calcification medium upregulated key calcification markers in ECs. HCAECs cultured and treated with calcification medium (CM) containing 2.0 mM HNa 2 PO 4 and 2.7 mM CaCl 2 for 24 h, in normoxia (37 °C humidified atmosphere with 5% CO 2 ). RNA was extracted using Trizol, and RNA was reverse-transcribed into cDNA using iSCRIPT buffer. ALP assay (Wako, Lab Assay) was used to quantify ALP levels in cell culture supernatants following CM treatment. Real-time PCR was performed using universal SYBR © Green Supermix. Relative changes in mRNA expression were calculated using the ∆∆Ct method. Whole-cell lysates were extracted to measure total and phosphorylated protein expression via immunoblotting technique. Even protein loading was confirmed by α-tubulin. ( A ) Molecular pathways for vascular calcification, ( B ) ALP activity, ( C ) BMP2 mRNA expression, ( D ) Runx2 mRNA expression, ( E ) BMP2 protein expression, ( F ) Runx2 protein expression. All data are represented as mean ± SEM; Student’s unpaired t -test, ( n = 3/4), * p < 0.05, 0.01, *** p < 0.001. BMP2: bone morphogenetic protein 2, Runx2: runt-related transcription factor, ALP: alkaline phosphatase.

Article Snippet: Membranes were probed with antibodies for BMP2 (NBP119751, Novus Biologicals, Centennial, CO, USA, 1:1000), total HIF-1α (NB100–449, Novus Biologicals, 1:500), VEGFA (ab461154, Abcam, Trumpington, Cambridge, UK, 1:2000), total VEGFR2 (2479; Cell Signalling Technology, Danvers, MA, USA, 1:1000), phosphorylated (y 1175 ) VEGFR2 (2478, Cell Signalling Technology, 1:1000), total eNOS (610297, BD Biosciences, San Jose, CA, USA, 1:1000), and phosphorylated (S 1177 ) eNOS (61293, BD Biosciences, 1:1000).

Techniques: Cell Culture, Reverse Transcription, ALP Assay, Real-time Polymerase Chain Reaction, SYBR Green Assay, Expressing, Western Blot, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Pro-Calcific Environment Impairs Ischaemia-Driven Angiogenesis

doi: 10.3390/ijms23063363

Figure Lengend Snippet: Pro-calcific environment differentially regulated gene expression of calcification and angiogenic genes in ischaemic limbs OF 8-week-old OPG −/− mice. Femoral artery ligation was performed on 8-week-old male C57 BL/6 mice (controls) and OPG −/− mice ( n = 5/group). At day 14, mice were culled, and gastrocnemius tissues were harvested for RNA. Real-time PCR was performed to assess gene expression of key angiogenic markers in the gastrocnemius muscle of both C57 BL/6 and OPG −/− mice. mRNA data are represented as non- ischemic (Non-Isc) for control hindlimb, and ischemic (Isc) for surgical hindlimb. ( A ) Blood ALP levels, ( B ) Runx2, ( C ) BMP2, ( D ) Siah1, ( E ) Siah2, ( F ) PHD1 mRNA expression, ( G ) PHD2, ( H ) HIF-1α ( I ) VEGFA, ( J ) VEGFR2. All data are represented as percentage of controls, mean ± SEM; * p < 0.05, ** p < 0.01, *** p <0.001, **** p < 0.0001, unpaired t -test, ( n = 3–5). HIF-1α, hypoxia-inducible factor-1 alpha; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2; PHD1/2, propyl hydroxylase domains 1/2; Runx2: runt related transcription factor; BMP2: bone morphogenetic protein 2; OPG: osteoprotegerin.

Article Snippet: Membranes were probed with antibodies for BMP2 (NBP119751, Novus Biologicals, Centennial, CO, USA, 1:1000), total HIF-1α (NB100–449, Novus Biologicals, 1:500), VEGFA (ab461154, Abcam, Trumpington, Cambridge, UK, 1:2000), total VEGFR2 (2479; Cell Signalling Technology, Danvers, MA, USA, 1:1000), phosphorylated (y 1175 ) VEGFR2 (2478, Cell Signalling Technology, 1:1000), total eNOS (610297, BD Biosciences, San Jose, CA, USA, 1:1000), and phosphorylated (S 1177 ) eNOS (61293, BD Biosciences, 1:1000).

Techniques: Gene Expression, Ligation, Real-time Polymerase Chain Reaction, Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Pro-Calcific Environment Impairs Ischaemia-Driven Angiogenesis

doi: 10.3390/ijms23063363

Figure Lengend Snippet: Summary schematic illustrating the effects of calcification on ischaemia-driven angiogenesis. Runx2: runt-related transcription factor, BMP2: bone morphogenetic protein, OPG: osteoprotegerin, ALP: alkaline phosphatase, PHD1/2: propyl hydroxylase domains 1/2, HIF-1 α : hypoxia-inducible factor-1 alpha, VEGFA: vascular endothelial growth factor, VEGFR2: vascular endothelial growth factor receptor 2, RANKL: receptor activator of nuclear factor ĸB ligand, RANK: receptor activator of nuclear factor κB, eNOS: endothelial nitric oxide synthase, HLI: hind-limb ischaemia.

Article Snippet: Membranes were probed with antibodies for BMP2 (NBP119751, Novus Biologicals, Centennial, CO, USA, 1:1000), total HIF-1α (NB100–449, Novus Biologicals, 1:500), VEGFA (ab461154, Abcam, Trumpington, Cambridge, UK, 1:2000), total VEGFR2 (2479; Cell Signalling Technology, Danvers, MA, USA, 1:1000), phosphorylated (y 1175 ) VEGFR2 (2478, Cell Signalling Technology, 1:1000), total eNOS (610297, BD Biosciences, San Jose, CA, USA, 1:1000), and phosphorylated (S 1177 ) eNOS (61293, BD Biosciences, 1:1000).

Techniques: