Journal: Clinical Cancer Research

Article Title: Vascular Endothelial Growth Factor Receptors VEGFR-2 and VEGFR-3 Are Localized Primarily to the Vasculature in Human Primary Solid Cancers

doi: 10.1158/1078-0432.ccr-09-2797

Figure Lengend Snippet: Fig. 1. Specificity of VEGFR-2 and VEGFR-3 antibodies. A, the specificity of antibodies raised to VEGFR-2 (55B11, sc-315, sc-504, and sc-6251) and VEGFR-3 (AF349) was determined by Western blot analysis of protein extracts from PAE and NIH-3T3 cells engineered to overexpress VEGFR-1, VEGFR-2, VEGFR-3, and CSF1R and cells expressing endogenous PDGFR-β (VSMC) and c-Kit (M-07e). B, 55B11 and AF349 were evaluated by immunocytochemical analysis of FFPE PAE cells engineered to overexpress VEGFRs.

Article Snippet: To do this, we determined the crossreactivity of human VEGFR antibodies using multiple www.aacrjournals.org Researc on Januarclincancerres.aacrjournals.org Downloaded from screening platforms and identified two antibodies, 55B11 (VEGFR-2; Cell Signaling Technology) and AF349 (VEGFR-3; R&D Systems), with credible specificity.

Techniques: Western Blot, Expressing

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: List of the analyzed ECM components.

Article Snippet: FLT4/VEGF3 , FLT4-Hs00176607_m1.

Techniques:

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: Invasion spectrum (the mRNA expression pattern of invasion-associated extracellular matrix components) differs in patients with ‘worse’ and ‘better’ prognoses. mRNA expression measurements were performed twice for each gene to confirm the data. A longer bar on the logarithmic scale indicates reduced expression. *P<0.05 vs. group A (Mann-Whitney U test). Group A, OS <24 months; group B, OS >24 months; BCAN, brevican; CD44, cluster of differentiation 44; CSPG5, chondroitin sulfate proteoglycan 5; EGFR, epidermal growth factor receptor; FLT4, Fms-related tyrosine kinase 4; HMMR, hyaluronan-mediated motility receptor; IDH1, isocitrate dehydrogenase 1; ITGAV, integrin-αV; MDM2, murine double minute 2; MMP-2, matrix metallopeptidase 2; NCAN, neurocan; PDGFA, platelet-derived growth factor α; TNC, tenascin C; VCAN, versican; OS, overall survival.

Article Snippet: FLT4/VEGF3 , FLT4-Hs00176607_m1.

Techniques: Expressing, MANN-WHITNEY, Derivative Assay

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: A total of 3 invasion-associated extracellular matrix components demonstrated significantly different expression levels in the samples from different prognostic groups. All 3 molecules had increased expression in patients in group A, indicating that the level of these molecules was associated with tumor invasiveness and patient survival. Group A, OS <24 months; group B, OS >24 month.; FLT4, Fms-related tyrosine kinase 4; MDM2, murine double minute 2; MMP-2, matrix metallopeptidase 2; rel., relative; OS, overall survival.

Article Snippet: FLT4/VEGF3 , FLT4-Hs00176607_m1.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: Immunohistochemical images of glioblastoma stained for FLT4/VEGF-3. (A) Overexpression of FLT4/VEGF-3 in patients with glioblastoma with a worse prognosis. (B) Moderate positivity in patients with a better prognosis (staining is notably reduced compared with that in patients with a worse prognosis). (C) Negative control. All images depicted are at ×20 magnification. FLT4, Fms-related tyrosine kinase 4; VEGF, vascular endothelial growth factor.

Article Snippet: FLT4/VEGF3 , FLT4-Hs00176607_m1.

Techniques: Immunohistochemical staining, Staining, Over Expression, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Expression levels of VEGF-C and VEGFR-3 in renal cell carcinoma and their association with lymph node metastasis

doi: 10.3892/etm.2021.9986

Figure Lengend Snippet: Immunohistochemical staining of VEGFR-3 expression. VEGFR-3 immunohistochemical staining in (A) normal renal tissues and (B) RCC tissues. (C) Lymphatic endothelial cells in RCC tissues were VEGFR-3 + (indicated by arrows). Scale bars, 100 µm. (D) Correlation between VEGF-C and VEGFR-3 expression in RCC tissues. Statistical significance was determined by Spearman's correlation test. RCC, renal cell carcinoma; VEGF-C, vascular endothelial growth factor-C; VEGFR-3, VEGF receptor-3.

Article Snippet: For antigen retrieval, the tissue sections were incubated with 0.01 M sodium citrate (pH 6) in a microwave oven at 95˚C for 10 min, followed by blocking with 5% normal goat serum (cat. no. ZLI-9021; OriGene Technologies, Inc.) for 10 min at room temperature, the tissue sections were incubated with rabbit anti-human VEGF-C monoclonal antibody (cat. no. BA0548; 1:200; Wuhan Boster Biological Technology Co., Ltd.), rabbit anti-human VEGFR-3 monoclonal antibody (cat. no. A01276-3; 1:200; Wuhan Boster Biological Technology Co., Ltd.) and mouse anti-human D2-40 monoclonal antibody (cat. no. ZM-0465; undiluted; OriGene Technologies, Inc.) for 12 h at 4˚C.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Science Advances

Article Title: CRISPR-based gene editing enables FOXP3 gene repair in IPEX patient cells

doi: 10.1126/sciadv.aaz0571

Figure Lengend Snippet: ( A ) Flow cytometry time course showing kinetics of FOXP3 expression in nonactivated T eff cells and activated T eff cells on subsequent days after activation [day 3 (d3), d6, and d14], comparing WT unmodified, WT mock-treated, and FOXP3 FLcoW edited T eff cells. FOXP3 expression quantified in T eff cells over time course of activation, showing average MFI (mean ± SD, n = 3). ( B ) Cytokine production in WT and FOXP3 gene edited T eff cells determined by enzyme-linked immunosorbent assay (ELISA). Supernatants collected at 24 hours (IL-2) and 48 hours (IFN-γ and IL-17) after activation with anti-CD3/28 (mean ± SD, n = 3). ( C ) T eff cell proliferation in response to activation measured by the proliferation assay. Flow cytometry plots of carboxyfluorescein diacetate succinimidyl ester (CFSE) dye–stained T eff cells with progressive dilution of dye as proliferation progresses from nonactivated to day 2 and day 3 after activation with anti-CD3/28 Dynabeads are shown. Comparison of proliferation rates in response to activation with a bead:cell ratio of 1:100 and 1:25. Quantification of average proliferative response of T eff cells from proliferation assay at day 3 is shown to the right, comparing different doses of activation beads (mean ± SD, n = 3).

Article Snippet: Both cell fractions were activated with immobilized plate-bound anti-CD3 [10 μg/ml; OKT3 monoclonal antibody (mAb), Miltenyi] with soluble anti-CD28 mAb (1 μg/ml; NA/LE, BD) for 2 to 3 days before editing and then switched to nonactivation conditions.

Techniques: Flow Cytometry, Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Staining, Comparison

Journal: Science Advances

Article Title: CRISPR-based gene editing enables FOXP3 gene repair in IPEX patient cells

doi: 10.1126/sciadv.aaz0571

Figure Lengend Snippet: ( A ) Differentiation potential of edited HSPCs tested by the in vitro CFU assay. Four resulting hematopoietic progenitor colony types: CFU-E (mature erythroid progenitors), CFU-GEMM (granulocyte, erythrocyte, macrophage, and megakaryocyte), BFU-E (primitive erythroid progenitors), and CFU-GM (granulocyte and macrophage progenitors). Representative images of colonies from the CFU assay, showing similar morphology (×10 magnification). ( B ) Experimental timeline of hu-mouse study using NSG-SGM3 mice. ( C ) Human engraftment kinetics in the peripheral blood of hu-mice at corresponding weeks after injection. Engraftment was measured by flow cytometry for hCD45 marker on human cells, and frequency was quantified relative to the total of human (hCD45 + ) and mouse (mCD45 + ) cells (mean ± SD). ( D ) Representative flow cytometry plots of engrafted human hematopoietic subsets in the bone marrow (left) and spleen (right) of hu-mice at 14 weeks after injection. Populations gated out of human cells (hCD45 + ). FOXP3 edited samples were divided into tNGFR + and tNGFR − gates for comparability. ( E ) Quantification of human hematopoietic lineages by flow cytometry with each symbol representing a single mouse (mean ± SD). In spleen, the CD8 + , CD4 + , and CD4 + CD8 + double-positive (DP) populations were gated out of CD3 + T cells. The CD25 + FOXP3 + , naïve CD45RA + , and memory CD45RA − populations were gated out of CD4 + single-positive T cell subset (* P < 0.5, ** P < 0.01, *** P < 0.001).

Article Snippet: Both cell fractions were activated with immobilized plate-bound anti-CD3 [10 μg/ml; OKT3 monoclonal antibody (mAb), Miltenyi] with soluble anti-CD28 mAb (1 μg/ml; NA/LE, BD) for 2 to 3 days before editing and then switched to nonactivation conditions.

Techniques: In Vitro, Colony-forming Unit Assay, Injection, Flow Cytometry, Marker

Journal: RSC Advances

Article Title: Discovery of VEGFR2 inhibitors by integrating naïve Bayesian classification, molecular docking and drug screening approaches †

doi: 10.1039/c7ra12259d

Figure Lengend Snippet: Workflow of the integrated virtual screening and drug screening approaches for VEGFR2 kinase inhibitors.

Article Snippet: Human VEGFR2 (cytoplastic domain expressed as N-terminal GST-fusion protein) was purchased from Carna Biosciences (Kobe, Japan); The HTRF® KinEASETM-TK assay kit (62TK0PEB) was provided by Cisbio Bioassays (Parc Marcel Boiteux, 30200 Codolet, France).

Techniques:

Journal: RSC Advances

Article Title: Discovery of VEGFR2 inhibitors by integrating naïve Bayesian classification, molecular docking and drug screening approaches †

doi: 10.1039/c7ra12259d

Figure Lengend Snippet: Examples of the top 15 good (A) and bad (B) fragments for VEGFR2 inhibition as estimated by NB-c model. The Bayesian score (Score) is given for each fragment.

Article Snippet: Human VEGFR2 (cytoplastic domain expressed as N-terminal GST-fusion protein) was purchased from Carna Biosciences (Kobe, Japan); The HTRF® KinEASETM-TK assay kit (62TK0PEB) was provided by Cisbio Bioassays (Parc Marcel Boiteux, 30200 Codolet, France).

Techniques: Inhibition

Journal: RSC Advances

Article Title: Discovery of VEGFR2 inhibitors by integrating naïve Bayesian classification, molecular docking and drug screening approaches †

doi: 10.1039/c7ra12259d

Figure Lengend Snippet: EstPGood values and LibDock Scores of FDA-approved drugs as predicted by NB-c and LibDock. While the 8 VEGFR2-targeting antiangiogenic agents are represented in red triangles, other drugs are represented in blue squares.

Article Snippet: Human VEGFR2 (cytoplastic domain expressed as N-terminal GST-fusion protein) was purchased from Carna Biosciences (Kobe, Japan); The HTRF® KinEASETM-TK assay kit (62TK0PEB) was provided by Cisbio Bioassays (Parc Marcel Boiteux, 30200 Codolet, France).

Techniques:

Journal: RSC Advances

Article Title: Discovery of VEGFR2 inhibitors by integrating naïve Bayesian classification, molecular docking and drug screening approaches †

doi: 10.1039/c7ra12259d

Figure Lengend Snippet: Inhibitory curves and IC 50 values for the reference compound axitinib (A) and three FDA-approved drugs flubendazole (B), rilpivirine (C), papaverine (D) against VEGFR2.

Article Snippet: Human VEGFR2 (cytoplastic domain expressed as N-terminal GST-fusion protein) was purchased from Carna Biosciences (Kobe, Japan); The HTRF® KinEASETM-TK assay kit (62TK0PEB) was provided by Cisbio Bioassays (Parc Marcel Boiteux, 30200 Codolet, France).

Techniques:

Journal: RSC Advances

Article Title: Discovery of VEGFR2 inhibitors by integrating naïve Bayesian classification, molecular docking and drug screening approaches †

doi: 10.1039/c7ra12259d

Figure Lengend Snippet: The receptor–ligand interactions of the axitinib (A, B), flubendazole (C, D), rilpivirine (E, F), papaverine (G, H) with the active site of VEGFR2.

Article Snippet: Human VEGFR2 (cytoplastic domain expressed as N-terminal GST-fusion protein) was purchased from Carna Biosciences (Kobe, Japan); The HTRF® KinEASETM-TK assay kit (62TK0PEB) was provided by Cisbio Bioassays (Parc Marcel Boiteux, 30200 Codolet, France).

Techniques:

Journal:

Article Title: A model for gene therapy of human hereditary lymphedema

doi: 10.1073/pnas.221449198

Figure Lengend Snippet: The VEGFR-3(I1053F) mutant is tyrosine kinase inactive. (A) The alignment of the human and mouse VEGFR-3 amino acid sequences showing the lymphedema-linked human mutations (bold) and the I1053F mutation found in the Chy mice (red). (B) Localization of the I1053F mutation within the catalytic domain of the ligand-bound VEGFR-3 heterodimer. (C) VEGFR-3(I1053F) is kinase inactive. We transfected cells with Mock, WT, or I1053F VEGFR-3 expression vectors and analyzed VEGFR-3 by immunoprecipitation and Western blotting of the cell lysates using phosphotyrosine antibodies (Upper). We also confirmed the expression of similar amounts of VEGFR-3 (Lower).

Article Snippet: We used the biotinylated anti-mouse VEGFR-3 Ab (R&D Systems, Oxon, U.K.) for whole-mount stainings.

Techniques: Mutagenesis, Transfection, Expressing, Immunoprecipitation, Western Blot

Journal:

Article Title: A model for gene therapy of human hereditary lymphedema

doi: 10.1073/pnas.221449198

Figure Lengend Snippet: Defective lymphatic vessels in the Chy mice. (A and B) The Chy mice were recognized by the accumulation of chyous fluid into the abdomen (red arrow). WT littermate is shown as a comparison. The stomach is marked with a dashed line. (C–F) The subserosal lymphatic vessels are enlarged in the Chy mouse intestine as detected by VEGFR-3 IHC (C and D), and by VEGFR-3 wholemount staining (E and F). (G) The feet of the Chy mice are swollen (arrows), when compared with a WT littermate. (H and I) MRI of the mouse feet shows the prominent hyperintensity in the Chy mouse foot, which is absent in a WT mouse. (J and K) The lymphatic endothelium (black arrows) of E15.5 skin was visualized by VEGFR-3 IHC in the Chy and WT mice. Note the absence of the lymphatic vessels in the Chy mice. (L and M) PECAM-1 IHC reveals no differences in the blood vasculature (red arrows). (N and O) VEGFR-3 IHC indicates that the lymphatics surrounding the aorta (a) in the Chy mice are similar to those of the WT mice. (P and Q) The hematoxylin-eosin staining of the back skin shows that the dermis (d) and s.c. adipose tissue (sc) are thickened in the Chy mice when compared with the WT littermate. (R and S) The transport of the Evans blue dye into the collecting lymphatic vessels (arrow) was visualized in the WT but not in the Chy mice after intradermal injection of the dye. [Bars = 5 mm (A and B), 90 μm (C and D), 200 μm (E and F), 5 mm (G), 100 μm (H and I), 70 μm (J–M), 200 μm (N and O), 210 μm (P and Q), and 1.5 mm (R and S).]

Article Snippet: We used the biotinylated anti-mouse VEGFR-3 Ab (R&D Systems, Oxon, U.K.) for whole-mount stainings.

Techniques: Comparison, Staining, Injection

Journal:

Article Title: A model for gene therapy of human hereditary lymphedema

doi: 10.1073/pnas.221449198

Figure Lengend Snippet: NRP-2 binds VEGF-C and is differentially expressed in the visceral organs and in the skin. (A) Labeled VEGF-C is precipitated by VEGFR-3-IgG and by NRP-2-IgG, but not by VEGFR-1-IgG fusion protein. (B and C) The intestinal VEGFR-3-positive lymphatic vessels (C; black arrow) stain also for NRP-2 (B), whereas the blood vessels are not stained (red arrows). (D and E) The VEGFR-3-positive vessels in the skin (E) are not stained by the NRP-2 antibodies (D). (Bars = 35 μm.)

Article Snippet: We used the biotinylated anti-mouse VEGFR-3 Ab (R&D Systems, Oxon, U.K.) for whole-mount stainings.

Techniques: Labeling, Staining

Journal:

Article Title: A model for gene therapy of human hereditary lymphedema

doi: 10.1073/pnas.221449198

Figure Lengend Snippet: Gene therapy by using viral VEGF-C overexpression. (A and B) Adenovirally induced VEGF-C overexpression in a Chy mouse ear induces formation of functional lymphatic vessels, as analyzed by fluorescent microlymphography (A) or by VEGFR-3 IHC (B). The dye depot is marked with a dashed line. (C and D) Microlymphography of the lymphatic vessels 7 weeks after AAV infection reveals functional lymphatic vessels (white arrows) in the AAV-VEGF-C-infected Chy ear (C) when compared with the control ear (D). (E and F) VEGFR-3 IHC shows that the AAV-VEGF-C-infected Chy ears contain VEGFR-3-positive vessels (E, black arrows), whereas no staining is detected in the AAV-EGFP-infected ears (F). (G and H) The fluorescent dextran was collected by the draining lymphatic vessel (white arrow) in the AAV-VEGF-C-treated Chy ear (G), unlike in the noninfected ear of the same mouse (H). Red arrows mark the blood vessels, and an arrowhead marks the cartilage of the ear. (I and J) In WT mice, there are no major changes in lymphatic vasculature after AAV-VEGF-C infection (I), when compared with the control ears (J). (K and L) The same is also confirmed by VEGFR-3 IHC. [Bars = 200 μm (A), 150 μm (B, E, F, K, and L), 300 μm (C, D, I, and J), and 5 mm (G and H).]

Article Snippet: We used the biotinylated anti-mouse VEGFR-3 Ab (R&D Systems, Oxon, U.K.) for whole-mount stainings.

Techniques: Over Expression, Functional Assay, Infection, Staining

Journal:

Article Title: A model for gene therapy of human hereditary lymphedema

doi: 10.1073/pnas.221449198

Figure Lengend Snippet: Analysis of the AAV-VEGF-C expression. (A) AAV-produced VEGF-C polypeptides were metabolically labeled and subjected to immunoprecipitation with VEGF-C antibodies (IP) or to binding assay with soluble VEGFR-3 and NRP-2 IgG fusion proteins. AAV-EGFP-infected cells were used as a negative control and VEGF-C produced in 293T cells as a positive control (p-C). (B) Northern blotting of total RNA from AAV-VEGF-C-infected and control ears of a Chy mouse.

Article Snippet: We used the biotinylated anti-mouse VEGFR-3 Ab (R&D Systems, Oxon, U.K.) for whole-mount stainings.

Techniques: Expressing, Produced, Metabolic Labelling, Labeling, Immunoprecipitation, Binding Assay, Infection, Negative Control, Positive Control, Northern Blot

Journal:

Article Title: A model for gene therapy of human hereditary lymphedema

doi: 10.1073/pnas.221449198

Figure Lengend Snippet: Lymphatic vessel growth in the Chy × K14-VEGF-C156S mice. (A–C) Staining by using VEGFR-3 antibodies shows lymphatic vessels (black arrows) in the ear of the Chy × K14-VEGF-C156S mice, in comparison with the aplastic lymphatic vessels in the Chy mouse ear (B), or with the WT littermate (C). (D–F) PECAM IHC confirms that the blood vasculature is normal in all mice. (G–I) VEGF-C IHC shows the VEGF-C156S transgene expression in the basal cells of the hair follicles (red arrows). (J–L) Fluorescent microlymphography shows the functional lymphatic capillary network in the Chy × K14-VEGF-C156S ear (J), resembling that of the WT mouse (L). The FITC-dextran is not collected into the lymphatic vessels in the Chy mouse ear (K). [Bars = 70 μm (A–F), 25 μm (G–I), and 600 μm (J–L).]

Article Snippet: We used the biotinylated anti-mouse VEGFR-3 Ab (R&D Systems, Oxon, U.K.) for whole-mount stainings.

Techniques: Staining, Comparison, Expressing, Functional Assay