vascular endothelial growth factor vegf a Search Results


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  • 99
    Thermo Fisher vegf a
    <t>VEGF-A</t> is differentially expressed in AMs in vivo in response to <t>TSLP</t> hi vs. TSLP lo tumor settings. (A) Strategy shown for the experimental design and RNA sample selection for panel B. (B) VEGF-A expression as measured by qRT-PCR analysis. 100 ng/mL recombinant TSLP was added to the AM/4T1-KD co-culture system and gene expression was quantified. (C) VEGF-A expression of AMs in vivo collected from the indicated tumor-bearing mice at 7 (left), 14 (middle) or 28 (right) days post-intravenous injection. For B and C, data were normalized to the housekeeping gene GAPDH. Then one 4T1-VC sample in panel B (n = 3 biologic replicates) or one 4T1-VC sample from each time point in panel C was set to 1.0 to determine the relative expression of the other samples. Panel C represents a total of 9 separate mice per tumor-bearing group, covering the 3 distinct time points shown. Therefore, at each time point, 3 separate mice from each cohort were analyzed, and for each mouse, 3–4 technical replicates were collected. Results represent the mean ± SEM of all replicates for each tumor-bearing group at each time point. * P
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    Millipore vegf
    <t>VEGF-A</t> is differentially expressed in AMs in vivo in response to <t>TSLP</t> hi vs. TSLP lo tumor settings. (A) Strategy shown for the experimental design and RNA sample selection for panel B. (B) VEGF-A expression as measured by qRT-PCR analysis. 100 ng/mL recombinant TSLP was added to the AM/4T1-KD co-culture system and gene expression was quantified. (C) VEGF-A expression of AMs in vivo collected from the indicated tumor-bearing mice at 7 (left), 14 (middle) or 28 (right) days post-intravenous injection. For B and C, data were normalized to the housekeeping gene GAPDH. Then one 4T1-VC sample in panel B (n = 3 biologic replicates) or one 4T1-VC sample from each time point in panel C was set to 1.0 to determine the relative expression of the other samples. Panel C represents a total of 9 separate mice per tumor-bearing group, covering the 3 distinct time points shown. Therefore, at each time point, 3 separate mice from each cohort were analyzed, and for each mouse, 3–4 technical replicates were collected. Results represent the mean ± SEM of all replicates for each tumor-bearing group at each time point. * P
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    Santa Cruz Biotechnology vegf a
    MVs shed from breast cancer cells contain an oligomeric <t>VEGF</t> species. ( a ) Whole cell lysates (WCL) from MDAMB231 cells (lane 1) and human recombinant VEGF 165 (rVEGF 165 ; lane 2) were immunoblotted with antibodies against VEGF 165 . ( b ) Concentrated conditioned medium (20 μg total protein) from serum-starved MDAMB231 (lane 1), HeLa (lane 2) or SKBR3 cells (lane 3) were immunoblotted. ( c ) MDAMB231 cells were analysed by immunofluorescent (IF) microscopy using Rhodamine-conjugated phalloidin (top panels) and either a pan VEGF or anti-VEGF 165 antibody (middle panels). Arrows indicate VEGF localized on MVs. Scale bar, 2 μm. IF images of MDAMB231 cells stained by secondary antibody (control; bottom panels). Scale bar, 10 μm ( d ) Exosomes (lane 1) or MVs (lane 2) from MDAMB231 cells were isolated, lysed and immunoblotted (5 μg per samples) with antibodies against VEGF, CD-63, actin and flotillin-2. ( e ) MVs from SKBR3 (lane 2), HeLa (lane 3) or MDAMB231 (lane 4) cells were isolated and lysed. WCL from MDAMB231 cells (lane 1), as well as MV lysates (10 μg per sample), were immunoblotted with antibodies against VEGF 165 , flotillin-2 and the cytosolic-specific marker IκBα. ( f ) MVs from MDAMB231 cells transfected with control siRNA (lane 1) or siRNAs targeting VEGF (lanes 2–4) were immunoblotted with a pan VEGF antibody or anti-flotillin-2. WCL were immunoblotted with a pan VEGF antibody and an anti-actin antibody. ( g ) Tubulogenesis assays on HUVECs that were untreated (control) or treated with MVs from MDAMB231 cells (10 μg ml −1 MV protein) transfected with control siRNA or siRNAs targeting VEGF. ( h ) MVs from MDAMB231 cells transfected with control siRNA (lane 1) or siRNAs targeting tTG (lanes 2 and 3) or treated with MDC (50 μM) (lane 4) were immunoblotted with antibodies against VEGF or flotillin-2, while WCLs were immunoblotted with antibodies against VEGF, tTG or actin. ( i ) Tubulogenesis assays of HUVECs treated with MVs (10 μg ml −1 MV protein) from MDAMB231 cells expressing control siRNA, siRNAs targeting tTG or with MVs from MDAMB231 cells treated with 50 μM MDC.
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    R&D Systems vegf protein
    MVs shed from breast cancer cells contain an oligomeric <t>VEGF</t> species. ( a ) Whole cell lysates (WCL) from MDAMB231 cells (lane 1) and human recombinant VEGF 165 (rVEGF 165 ; lane 2) were immunoblotted with antibodies against VEGF 165 . ( b ) Concentrated conditioned medium (20 μg total protein) from serum-starved MDAMB231 (lane 1), HeLa (lane 2) or SKBR3 cells (lane 3) were immunoblotted. ( c ) MDAMB231 cells were analysed by immunofluorescent (IF) microscopy using Rhodamine-conjugated phalloidin (top panels) and either a pan VEGF or anti-VEGF 165 antibody (middle panels). Arrows indicate VEGF localized on MVs. Scale bar, 2 μm. IF images of MDAMB231 cells stained by secondary antibody (control; bottom panels). Scale bar, 10 μm ( d ) Exosomes (lane 1) or MVs (lane 2) from MDAMB231 cells were isolated, lysed and immunoblotted (5 μg per samples) with antibodies against VEGF, CD-63, actin and flotillin-2. ( e ) MVs from SKBR3 (lane 2), HeLa (lane 3) or MDAMB231 (lane 4) cells were isolated and lysed. WCL from MDAMB231 cells (lane 1), as well as MV lysates (10 μg per sample), were immunoblotted with antibodies against VEGF 165 , flotillin-2 and the cytosolic-specific marker IκBα. ( f ) MVs from MDAMB231 cells transfected with control siRNA (lane 1) or siRNAs targeting VEGF (lanes 2–4) were immunoblotted with a pan VEGF antibody or anti-flotillin-2. WCL were immunoblotted with a pan VEGF antibody and an anti-actin antibody. ( g ) Tubulogenesis assays on HUVECs that were untreated (control) or treated with MVs from MDAMB231 cells (10 μg ml −1 MV protein) transfected with control siRNA or siRNAs targeting VEGF. ( h ) MVs from MDAMB231 cells transfected with control siRNA (lane 1) or siRNAs targeting tTG (lanes 2 and 3) or treated with MDC (50 μM) (lane 4) were immunoblotted with antibodies against VEGF or flotillin-2, while WCLs were immunoblotted with antibodies against VEGF, tTG or actin. ( i ) Tubulogenesis assays of HUVECs treated with MVs (10 μg ml −1 MV protein) from MDAMB231 cells expressing control siRNA, siRNAs targeting tTG or with MVs from MDAMB231 cells treated with 50 μM MDC.
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    Abcam vegf a
    MVs shed from breast cancer cells contain an oligomeric <t>VEGF</t> species. ( a ) Whole cell lysates (WCL) from MDAMB231 cells (lane 1) and human recombinant VEGF 165 (rVEGF 165 ; lane 2) were immunoblotted with antibodies against VEGF 165 . ( b ) Concentrated conditioned medium (20 μg total protein) from serum-starved MDAMB231 (lane 1), HeLa (lane 2) or SKBR3 cells (lane 3) were immunoblotted. ( c ) MDAMB231 cells were analysed by immunofluorescent (IF) microscopy using Rhodamine-conjugated phalloidin (top panels) and either a pan VEGF or anti-VEGF 165 antibody (middle panels). Arrows indicate VEGF localized on MVs. Scale bar, 2 μm. IF images of MDAMB231 cells stained by secondary antibody (control; bottom panels). Scale bar, 10 μm ( d ) Exosomes (lane 1) or MVs (lane 2) from MDAMB231 cells were isolated, lysed and immunoblotted (5 μg per samples) with antibodies against VEGF, CD-63, actin and flotillin-2. ( e ) MVs from SKBR3 (lane 2), HeLa (lane 3) or MDAMB231 (lane 4) cells were isolated and lysed. WCL from MDAMB231 cells (lane 1), as well as MV lysates (10 μg per sample), were immunoblotted with antibodies against VEGF 165 , flotillin-2 and the cytosolic-specific marker IκBα. ( f ) MVs from MDAMB231 cells transfected with control siRNA (lane 1) or siRNAs targeting VEGF (lanes 2–4) were immunoblotted with a pan VEGF antibody or anti-flotillin-2. WCL were immunoblotted with a pan VEGF antibody and an anti-actin antibody. ( g ) Tubulogenesis assays on HUVECs that were untreated (control) or treated with MVs from MDAMB231 cells (10 μg ml −1 MV protein) transfected with control siRNA or siRNAs targeting VEGF. ( h ) MVs from MDAMB231 cells transfected with control siRNA (lane 1) or siRNAs targeting tTG (lanes 2 and 3) or treated with MDC (50 μM) (lane 4) were immunoblotted with antibodies against VEGF or flotillin-2, while WCLs were immunoblotted with antibodies against VEGF, tTG or actin. ( i ) Tubulogenesis assays of HUVECs treated with MVs (10 μg ml −1 MV protein) from MDAMB231 cells expressing control siRNA, siRNAs targeting tTG or with MVs from MDAMB231 cells treated with 50 μM MDC.
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    Santa Cruz Biotechnology anti vegf a
    ( A ) Heart’s specimen from normal mice obtained 7 day after exposure to hypoxia. The expression of <t>VEGF</t> returns to initial levels. ( B ) Heart’s specimen from mdx mice obtained 7 day after exposure to hypoxia. The expression of VEGF in myocardium remains elevated but returns to normal in vessel walls. Scale bar =100 μm.
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    PeproTech vegf a
    FLI1 and PKC co-activation mediated hESCs differentiation into iECs a Schematic illustration of the EC differentiation strategy from hESCs. b Typical morphological images of hESC-EC differentiation on days of 0, 1, 2, and 3. Scale bar, 100 μm. c The ratio of CD31+/CD144+ cells gradually increased during induction. Columns represent the mean ± SD; n = 5 independent differentiation experiments. d Representative results of the percentage of CD31+/CD144+ cells during the induction process detected by FCM. e Overexpression of FLI1 and activation of PKC in different hESC lines (hESC-254 or hESC-137) induced iECs. f Overexpressing FLI1 with different PKC activators (PMA or prostratin) yielded iECs. g Expression levels of <t>VEGF</t> , GATA2 , CD31 and CD144 genes in hESCs, human fibroblasts (HFs), iECs and EPCs. Columns represent the mean ± SD; n = 5 independent differentiation experiments
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    SLIT2 LTD vegf
    FLI1 and PKC co-activation mediated hESCs differentiation into iECs a Schematic illustration of the EC differentiation strategy from hESCs. b Typical morphological images of hESC-EC differentiation on days of 0, 1, 2, and 3. Scale bar, 100 μm. c The ratio of CD31+/CD144+ cells gradually increased during induction. Columns represent the mean ± SD; n = 5 independent differentiation experiments. d Representative results of the percentage of CD31+/CD144+ cells during the induction process detected by FCM. e Overexpression of FLI1 and activation of PKC in different hESC lines (hESC-254 or hESC-137) induced iECs. f Overexpressing FLI1 with different PKC activators (PMA or prostratin) yielded iECs. g Expression levels of <t>VEGF</t> , GATA2 , CD31 and CD144 genes in hESCs, human fibroblasts (HFs), iECs and EPCs. Columns represent the mean ± SD; n = 5 independent differentiation experiments
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    R&D Systems recombinant human vegf a
    Assessment of <t>VEGF-A-induced</t> cell proliferation and migration upon modulation of miR-424–5p or miR-29a-3p. ( A ) Cell proliferation assessed by cytofluorimetric analysis of EdU incorporation into the DNA in control spheroids (SPHC) or VEGF-A-stimulated spheroids (SPHV) generated with ECs transfected with miR-424–5p or miR-29a-3p mimics or inhibitors or their respective controls. Representative plots for n = 3 experiments. ( B ) Cell proliferation assessed by cytofluorimetric analysis of EdU incorporation into the DNA during cell replication in 2D cultured HUVECs transfected with miR-424–5p or miR-29a-3p mimics or inhibitors, or respective transfection controls. The assay was performed after 18 hr of VEGF stimulation. Data are represented as mean ± SEM from n = 3 experiments. ( C ) VEGF-induced cell migration of HUVECs transfected with miR-424–5p or miR-29a-3p mimics or inhibitors, or respective transfection controls, assessed by xCELLigence and represented as the slope of the migration curve over a 24 hr experiment. Data are represented as mean ± SEM from n = 3 experiments. ***, p
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    R&D Systems vegf 165
    Assessment of <t>VEGF-A-induced</t> cell proliferation and migration upon modulation of miR-424–5p or miR-29a-3p. ( A ) Cell proliferation assessed by cytofluorimetric analysis of EdU incorporation into the DNA in control spheroids (SPHC) or VEGF-A-stimulated spheroids (SPHV) generated with ECs transfected with miR-424–5p or miR-29a-3p mimics or inhibitors or their respective controls. Representative plots for n = 3 experiments. ( B ) Cell proliferation assessed by cytofluorimetric analysis of EdU incorporation into the DNA during cell replication in 2D cultured HUVECs transfected with miR-424–5p or miR-29a-3p mimics or inhibitors, or respective transfection controls. The assay was performed after 18 hr of VEGF stimulation. Data are represented as mean ± SEM from n = 3 experiments. ( C ) VEGF-induced cell migration of HUVECs transfected with miR-424–5p or miR-29a-3p mimics or inhibitors, or respective transfection controls, assessed by xCELLigence and represented as the slope of the migration curve over a 24 hr experiment. Data are represented as mean ± SEM from n = 3 experiments. ***, p
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    Genentech vegf a
    Cellular proliferation, matrix metalloproteinase 2 ( MMP-2 ), and matrix metalloproteinase 9 ( MMP-9 ) are decreased in lentivirus (LV)–small hairpin RNA (shRNA)–vascular endothelial growth factor-A ( <t>VEGF-A</t> )–transduced vessels. ( a , upper panel) Representative sections after Ki-67 staining at the venous stenosis of the LV-shRNA- VEGF-A (LV) and scrambled shRNA (control (C)) or <t>Avastin-treated</t> vessels or immunoglobulin G (IgG) controls at days 14 (D14) and 28 (D28). Nuclei staining brown are positive for Ki-67. IgG antibody staining was performed to serve as negative controls. *Indicates the lumen. All are original magnification × 40. Bar=200 μm. Pooled data for the LV and C groups or Avastin-treated vessels or IgG controls are shown in the lower panel. This demonstrates a significant decrease in the mean Ki-67 index in the LV-transduced vessels when compared with the C vessels at day 14 ( P
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    R&D Systems vegf
    Pleiotropic effects of <t>NGF</t> on HUVEC . (A) Growth assay. HUVEC cultured on standard culture plastic were treated with NGF (100 ng/ml), or <t>VEGF</t> (10 ng/ml) for 48 h. (B and C) Migration and invasion assay using Transwells. (D) Cord formation assay on matrigel-coated 24-well dishes. (E) Endothelial cell monolayer permeability assay. The kinetics of 70 kDa Dextran-FITC having passed through the monolayer of endothelial cells was determined by measurement of fluorescence in the lower chambers at different time points. A to D Student's t-test, *p
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    Thermo Fisher gene exp vegfa hs00900055 m1
    Pleiotropic effects of <t>NGF</t> on HUVEC . (A) Growth assay. HUVEC cultured on standard culture plastic were treated with NGF (100 ng/ml), or <t>VEGF</t> (10 ng/ml) for 48 h. (B and C) Migration and invasion assay using Transwells. (D) Cord formation assay on matrigel-coated 24-well dishes. (E) Endothelial cell monolayer permeability assay. The kinetics of 70 kDa Dextran-FITC having passed through the monolayer of endothelial cells was determined by measurement of fluorescence in the lower chambers at different time points. A to D Student's t-test, *p
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    R&D Systems vegf protein levels
    Pleiotropic effects of <t>NGF</t> on HUVEC . (A) Growth assay. HUVEC cultured on standard culture plastic were treated with NGF (100 ng/ml), or <t>VEGF</t> (10 ng/ml) for 48 h. (B and C) Migration and invasion assay using Transwells. (D) Cord formation assay on matrigel-coated 24-well dishes. (E) Endothelial cell monolayer permeability assay. The kinetics of 70 kDa Dextran-FITC having passed through the monolayer of endothelial cells was determined by measurement of fluorescence in the lower chambers at different time points. A to D Student's t-test, *p
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    Reprokine rh vegf
    Pleiotropic effects of <t>NGF</t> on HUVEC . (A) Growth assay. HUVEC cultured on standard culture plastic were treated with NGF (100 ng/ml), or <t>VEGF</t> (10 ng/ml) for 48 h. (B and C) Migration and invasion assay using Transwells. (D) Cord formation assay on matrigel-coated 24-well dishes. (E) Endothelial cell monolayer permeability assay. The kinetics of 70 kDa Dextran-FITC having passed through the monolayer of endothelial cells was determined by measurement of fluorescence in the lower chambers at different time points. A to D Student's t-test, *p
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    Thermo Fisher vascular endothelial growth factor vegf a
    Pleiotropic effects of <t>NGF</t> on HUVEC . (A) Growth assay. HUVEC cultured on standard culture plastic were treated with NGF (100 ng/ml), or <t>VEGF</t> (10 ng/ml) for 48 h. (B and C) Migration and invasion assay using Transwells. (D) Cord formation assay on matrigel-coated 24-well dishes. (E) Endothelial cell monolayer permeability assay. The kinetics of 70 kDa Dextran-FITC having passed through the monolayer of endothelial cells was determined by measurement of fluorescence in the lower chambers at different time points. A to D Student's t-test, *p
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    Becton Dickinson vegf
    fVII is internalized by TF-expressing <t>VEGF-stimulated</t> angiogenic <t>HUVECs</t> and MDA-MB-231 breast cancer cells. fVII protein primarily bound to the cell plasma membrane ( green, arrows in a and d ) of the fixed cells (0 min), while endocytosis of fVII, shown as co-localization intracellularly with Alexa Fluor 633-phalloidin-stained cytoskeletal F-actin ( red, arrows in b and e ), was observed at 37°C for 30 min after incubation with VEGF-stimulated HUVECs ( b ) and human breast cancer MDA-MB-231 cells ( e ). c and f Secondary antibody FITC controls for non-specific FITC background. Scale bars , 20 µm
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    RayBiotech human vegf a elisa kit
    fVII is internalized by TF-expressing <t>VEGF-stimulated</t> angiogenic <t>HUVECs</t> and MDA-MB-231 breast cancer cells. fVII protein primarily bound to the cell plasma membrane ( green, arrows in a and d ) of the fixed cells (0 min), while endocytosis of fVII, shown as co-localization intracellularly with Alexa Fluor 633-phalloidin-stained cytoskeletal F-actin ( red, arrows in b and e ), was observed at 37°C for 30 min after incubation with VEGF-stimulated HUVECs ( b ) and human breast cancer MDA-MB-231 cells ( e ). c and f Secondary antibody FITC controls for non-specific FITC background. Scale bars , 20 µm
    Human Vegf A Elisa Kit, supplied by RayBiotech, used in various techniques. Bioz Stars score: 95/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit anti vegf a
    Fluorescence images of <t>VEGF</t> IR in detrusor smooth muscle in the bladder neck region in whole-mount preparations of urinary bladder in control rats ( A and B ) and rats treated with CYP for 4 h ( C and D ) and 48 h ( E and F ). VEGF IR in the detrusor smooth
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    R&D Systems recombinant vegf a
    Fluorescence images of <t>VEGF</t> IR in detrusor smooth muscle in the bladder neck region in whole-mount preparations of urinary bladder in control rats ( A and B ) and rats treated with CYP for 4 h ( C and D ) and 48 h ( E and F ). VEGF IR in the detrusor smooth
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    Thermo Fisher gene exp vegfa mm00437304 m1
    Fluorescence images of <t>VEGF</t> IR in detrusor smooth muscle in the bladder neck region in whole-mount preparations of urinary bladder in control rats ( A and B ) and rats treated with CYP for 4 h ( C and D ) and 48 h ( E and F ). VEGF IR in the detrusor smooth
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    Santa Cruz Biotechnology antibodies against vegf a
    Effects of <t>Lenti-miRNA-VEGF-A</t> and/or Lenti-miRNA-VEGF-C on cancer growth in GC bearing nude mice. A. Cancer volumes were significantly reduced in mice treated with Lenti-miRNA-VEGF-A, Lenti-miRNA-VEGF-C, or Lenti-miRNA-VEGF-A +VEGF-C, as compared to those
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    Thermo Fisher gene exp vegfa mm00437306 m1
    Effects of <t>Lenti-miRNA-VEGF-A</t> and/or Lenti-miRNA-VEGF-C on cancer growth in GC bearing nude mice. A. Cancer volumes were significantly reduced in mice treated with Lenti-miRNA-VEGF-A, Lenti-miRNA-VEGF-C, or Lenti-miRNA-VEGF-A +VEGF-C, as compared to those
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    Expression of Flag tag in hypoxic myocardium. The expression of Flag tag, which was fused with C-terminus of <t>VEGF,</t> was detected in ischemic myocardium by immunofluorescence staining. ( A ) Representative images for each condition is shown (scale bar = 50 μm). ( B ) The quantification of FITC fluorescence intensity was performed as described in Methods section. Results are presented as mean ± SEM (n = 8), ** p
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    Image Search Results


    VEGF-A is differentially expressed in AMs in vivo in response to TSLP hi vs. TSLP lo tumor settings. (A) Strategy shown for the experimental design and RNA sample selection for panel B. (B) VEGF-A expression as measured by qRT-PCR analysis. 100 ng/mL recombinant TSLP was added to the AM/4T1-KD co-culture system and gene expression was quantified. (C) VEGF-A expression of AMs in vivo collected from the indicated tumor-bearing mice at 7 (left), 14 (middle) or 28 (right) days post-intravenous injection. For B and C, data were normalized to the housekeeping gene GAPDH. Then one 4T1-VC sample in panel B (n = 3 biologic replicates) or one 4T1-VC sample from each time point in panel C was set to 1.0 to determine the relative expression of the other samples. Panel C represents a total of 9 separate mice per tumor-bearing group, covering the 3 distinct time points shown. Therefore, at each time point, 3 separate mice from each cohort were analyzed, and for each mouse, 3–4 technical replicates were collected. Results represent the mean ± SEM of all replicates for each tumor-bearing group at each time point. * P

    Journal: Oncoimmunology

    Article Title: Tumor-derived thymic stromal lymphopoietin enhances lung metastasis through an alveolar macrophage-dependent mechanism

    doi: 10.1080/2162402X.2017.1419115

    Figure Lengend Snippet: VEGF-A is differentially expressed in AMs in vivo in response to TSLP hi vs. TSLP lo tumor settings. (A) Strategy shown for the experimental design and RNA sample selection for panel B. (B) VEGF-A expression as measured by qRT-PCR analysis. 100 ng/mL recombinant TSLP was added to the AM/4T1-KD co-culture system and gene expression was quantified. (C) VEGF-A expression of AMs in vivo collected from the indicated tumor-bearing mice at 7 (left), 14 (middle) or 28 (right) days post-intravenous injection. For B and C, data were normalized to the housekeeping gene GAPDH. Then one 4T1-VC sample in panel B (n = 3 biologic replicates) or one 4T1-VC sample from each time point in panel C was set to 1.0 to determine the relative expression of the other samples. Panel C represents a total of 9 separate mice per tumor-bearing group, covering the 3 distinct time points shown. Therefore, at each time point, 3 separate mice from each cohort were analyzed, and for each mouse, 3–4 technical replicates were collected. Results represent the mean ± SEM of all replicates for each tumor-bearing group at each time point. * P

    Article Snippet: Protein levels were measured using an ELISA kit for TSLP, VEGF-A (both eBioscience, San Diego, CA) or G-CSF (RayBiotech, Norcross, GA).

    Techniques: Affinity Magnetic Separation, In Vivo, Selection, Expressing, Quantitative RT-PCR, Recombinant, Co-Culture Assay, Mouse Assay, Injection

    MVs shed from breast cancer cells contain an oligomeric VEGF species. ( a ) Whole cell lysates (WCL) from MDAMB231 cells (lane 1) and human recombinant VEGF 165 (rVEGF 165 ; lane 2) were immunoblotted with antibodies against VEGF 165 . ( b ) Concentrated conditioned medium (20 μg total protein) from serum-starved MDAMB231 (lane 1), HeLa (lane 2) or SKBR3 cells (lane 3) were immunoblotted. ( c ) MDAMB231 cells were analysed by immunofluorescent (IF) microscopy using Rhodamine-conjugated phalloidin (top panels) and either a pan VEGF or anti-VEGF 165 antibody (middle panels). Arrows indicate VEGF localized on MVs. Scale bar, 2 μm. IF images of MDAMB231 cells stained by secondary antibody (control; bottom panels). Scale bar, 10 μm ( d ) Exosomes (lane 1) or MVs (lane 2) from MDAMB231 cells were isolated, lysed and immunoblotted (5 μg per samples) with antibodies against VEGF, CD-63, actin and flotillin-2. ( e ) MVs from SKBR3 (lane 2), HeLa (lane 3) or MDAMB231 (lane 4) cells were isolated and lysed. WCL from MDAMB231 cells (lane 1), as well as MV lysates (10 μg per sample), were immunoblotted with antibodies against VEGF 165 , flotillin-2 and the cytosolic-specific marker IκBα. ( f ) MVs from MDAMB231 cells transfected with control siRNA (lane 1) or siRNAs targeting VEGF (lanes 2–4) were immunoblotted with a pan VEGF antibody or anti-flotillin-2. WCL were immunoblotted with a pan VEGF antibody and an anti-actin antibody. ( g ) Tubulogenesis assays on HUVECs that were untreated (control) or treated with MVs from MDAMB231 cells (10 μg ml −1 MV protein) transfected with control siRNA or siRNAs targeting VEGF. ( h ) MVs from MDAMB231 cells transfected with control siRNA (lane 1) or siRNAs targeting tTG (lanes 2 and 3) or treated with MDC (50 μM) (lane 4) were immunoblotted with antibodies against VEGF or flotillin-2, while WCLs were immunoblotted with antibodies against VEGF, tTG or actin. ( i ) Tubulogenesis assays of HUVECs treated with MVs (10 μg ml −1 MV protein) from MDAMB231 cells expressing control siRNA, siRNAs targeting tTG or with MVs from MDAMB231 cells treated with 50 μM MDC.

    Journal: Nature Communications

    Article Title: A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis

    doi: 10.1038/ncomms14450

    Figure Lengend Snippet: MVs shed from breast cancer cells contain an oligomeric VEGF species. ( a ) Whole cell lysates (WCL) from MDAMB231 cells (lane 1) and human recombinant VEGF 165 (rVEGF 165 ; lane 2) were immunoblotted with antibodies against VEGF 165 . ( b ) Concentrated conditioned medium (20 μg total protein) from serum-starved MDAMB231 (lane 1), HeLa (lane 2) or SKBR3 cells (lane 3) were immunoblotted. ( c ) MDAMB231 cells were analysed by immunofluorescent (IF) microscopy using Rhodamine-conjugated phalloidin (top panels) and either a pan VEGF or anti-VEGF 165 antibody (middle panels). Arrows indicate VEGF localized on MVs. Scale bar, 2 μm. IF images of MDAMB231 cells stained by secondary antibody (control; bottom panels). Scale bar, 10 μm ( d ) Exosomes (lane 1) or MVs (lane 2) from MDAMB231 cells were isolated, lysed and immunoblotted (5 μg per samples) with antibodies against VEGF, CD-63, actin and flotillin-2. ( e ) MVs from SKBR3 (lane 2), HeLa (lane 3) or MDAMB231 (lane 4) cells were isolated and lysed. WCL from MDAMB231 cells (lane 1), as well as MV lysates (10 μg per sample), were immunoblotted with antibodies against VEGF 165 , flotillin-2 and the cytosolic-specific marker IκBα. ( f ) MVs from MDAMB231 cells transfected with control siRNA (lane 1) or siRNAs targeting VEGF (lanes 2–4) were immunoblotted with a pan VEGF antibody or anti-flotillin-2. WCL were immunoblotted with a pan VEGF antibody and an anti-actin antibody. ( g ) Tubulogenesis assays on HUVECs that were untreated (control) or treated with MVs from MDAMB231 cells (10 μg ml −1 MV protein) transfected with control siRNA or siRNAs targeting VEGF. ( h ) MVs from MDAMB231 cells transfected with control siRNA (lane 1) or siRNAs targeting tTG (lanes 2 and 3) or treated with MDC (50 μM) (lane 4) were immunoblotted with antibodies against VEGF or flotillin-2, while WCLs were immunoblotted with antibodies against VEGF, tTG or actin. ( i ) Tubulogenesis assays of HUVECs treated with MVs (10 μg ml −1 MV protein) from MDAMB231 cells expressing control siRNA, siRNAs targeting tTG or with MVs from MDAMB231 cells treated with 50 μM MDC.

    Article Snippet: Reagents The pan VEGF antibody that recognizes all forms of VEGF-A was obtained from Santa Cruz (Dallas, TX, USA) (SC-507; used at 1:2,000).

    Techniques: Recombinant, Microscopy, Staining, Isolation, Marker, Transfection, Expressing

    Diagrams depicting cancer cells generating VEGF 90K and shedding MVs with associated VEGF 90K . ( a ) VEGF 165 is crosslinked by tTG to generate VEGF 90K (top). MVs with associated VEGF 90K are budding and shed from cancer cell plasma membrane (bottom). ( b ) Diagram depicting cancer cells shedding MVs with associated VEGF 90K that engage recipient endothelial cells and activate VEGFRs, thereby promoting angiogenesis. MV-associated Hsp90 binds to VEGF 90K , enabling the vesicles to activate VEGFRs on endothelial cells and stimulate the formation of new blood vessels. This stimulation is insensitive to Bevacizumab (top). 17AAG causes the release of VEGF 90K from MVs. The free VEGF 90K activates VEGFRs and stimulates angiogenesis but is sensitive to the inhibitory actions of Bevacizumab (bottom).

    Journal: Nature Communications

    Article Title: A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis

    doi: 10.1038/ncomms14450

    Figure Lengend Snippet: Diagrams depicting cancer cells generating VEGF 90K and shedding MVs with associated VEGF 90K . ( a ) VEGF 165 is crosslinked by tTG to generate VEGF 90K (top). MVs with associated VEGF 90K are budding and shed from cancer cell plasma membrane (bottom). ( b ) Diagram depicting cancer cells shedding MVs with associated VEGF 90K that engage recipient endothelial cells and activate VEGFRs, thereby promoting angiogenesis. MV-associated Hsp90 binds to VEGF 90K , enabling the vesicles to activate VEGFRs on endothelial cells and stimulate the formation of new blood vessels. This stimulation is insensitive to Bevacizumab (top). 17AAG causes the release of VEGF 90K from MVs. The free VEGF 90K activates VEGFRs and stimulates angiogenesis but is sensitive to the inhibitory actions of Bevacizumab (bottom).

    Article Snippet: Reagents The pan VEGF antibody that recognizes all forms of VEGF-A was obtained from Santa Cruz (Dallas, TX, USA) (SC-507; used at 1:2,000).

    Techniques:

    Reconstitution of Bevacizumab and 17AAG sensitivity. ( a ) Schematic of the in vivo angiogenesis assay. ( b , c ) Relative amounts of endothelial cells that entered implanted angioreactors that lacked any activators (vehicle control; histogram 1 in both graphs), or were loaded with MDAMB231 cells (5 × 10 4 cells per angioreactor) without ( b , histogram 2) or with a pan inactivating VEGF antibody (200 ng per angioreactor; b histogram 3) or were loaded with rVEGF 165 (4 ng per angioreactor) without ( c histogram 2) or with VEGF antibody (200 ng per angioreactor; C, histogram 3). ( d ) Scanning electron microscopy (s.e.m.) of a MDAMB231 cell. Arrows indicate large EVs. ( e ) MVs from MDAMB231 cells were examined by fluorescence staining using the membrane dye FM 1-43FX. Arrows indicate MVs. Scale bar, 5 μm. ( f ) MVs isolated from MDAMB231 cells were examined using rhodamine-conjugated phalloidin. Arrows indicate MVs. Scale bar, 5 μm. ( g ) The relative amounts of endothelial cells that entered angioreactors that lacked activators (control; histogram 1), contained MDAMB231 cell MVs (2 μg total protein per angioreactor) without (histogram 2) or with Bevacizumab (1 μg per angioreactor; histogram 3) or contained rVEGF 165 (4 ng per angioreactor) without (histogram 4) or with Bevacizumab (1 μg per angioreactor; histogram 5). ( h ) Relative amounts of endothelial cells that entered angioreactors that lacked activators (vehicle control; histogram 1), or angioreactors that contained the indicated combinations of MDAMB231 cell MVs (2 μg per angioreactor), 10 μM 17AAG and 1 μg Bevacizumab (histograms 2–4). ( i ) Tubulogenesis assays of HUVECs left untreated (control; histogram 1), treated with MVs (10 μg ml −1 total protein) from MDAMB231 cells without (histogram 2) or with 0.5 μg ml −1 Bevacizumab (histogram 3) or treated with rVEGF (15 ng ml −1 ) without (histogram 4) or with 0.5 μg ml −1 Bevacizumab (histogram 5). Left: The relative differences in tube lengths were plotted. Right: Images of the tubulogenesis assays performed under the different conditions tested. ( j ) Tubulogenesis assays were performed on HUVECs that were untreated (control; histogram 1) or treated with MVs (10 μg ml −1 of MV protein) from MDAMB231 cells pre-treated with various combinations of 10 μM 17AAG and 0.5 μg ml −1 Bevacizumab (histograms 2–5).

    Journal: Nature Communications

    Article Title: A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis

    doi: 10.1038/ncomms14450

    Figure Lengend Snippet: Reconstitution of Bevacizumab and 17AAG sensitivity. ( a ) Schematic of the in vivo angiogenesis assay. ( b , c ) Relative amounts of endothelial cells that entered implanted angioreactors that lacked any activators (vehicle control; histogram 1 in both graphs), or were loaded with MDAMB231 cells (5 × 10 4 cells per angioreactor) without ( b , histogram 2) or with a pan inactivating VEGF antibody (200 ng per angioreactor; b histogram 3) or were loaded with rVEGF 165 (4 ng per angioreactor) without ( c histogram 2) or with VEGF antibody (200 ng per angioreactor; C, histogram 3). ( d ) Scanning electron microscopy (s.e.m.) of a MDAMB231 cell. Arrows indicate large EVs. ( e ) MVs from MDAMB231 cells were examined by fluorescence staining using the membrane dye FM 1-43FX. Arrows indicate MVs. Scale bar, 5 μm. ( f ) MVs isolated from MDAMB231 cells were examined using rhodamine-conjugated phalloidin. Arrows indicate MVs. Scale bar, 5 μm. ( g ) The relative amounts of endothelial cells that entered angioreactors that lacked activators (control; histogram 1), contained MDAMB231 cell MVs (2 μg total protein per angioreactor) without (histogram 2) or with Bevacizumab (1 μg per angioreactor; histogram 3) or contained rVEGF 165 (4 ng per angioreactor) without (histogram 4) or with Bevacizumab (1 μg per angioreactor; histogram 5). ( h ) Relative amounts of endothelial cells that entered angioreactors that lacked activators (vehicle control; histogram 1), or angioreactors that contained the indicated combinations of MDAMB231 cell MVs (2 μg per angioreactor), 10 μM 17AAG and 1 μg Bevacizumab (histograms 2–4). ( i ) Tubulogenesis assays of HUVECs left untreated (control; histogram 1), treated with MVs (10 μg ml −1 total protein) from MDAMB231 cells without (histogram 2) or with 0.5 μg ml −1 Bevacizumab (histogram 3) or treated with rVEGF (15 ng ml −1 ) without (histogram 4) or with 0.5 μg ml −1 Bevacizumab (histogram 5). Left: The relative differences in tube lengths were plotted. Right: Images of the tubulogenesis assays performed under the different conditions tested. ( j ) Tubulogenesis assays were performed on HUVECs that were untreated (control; histogram 1) or treated with MVs (10 μg ml −1 of MV protein) from MDAMB231 cells pre-treated with various combinations of 10 μM 17AAG and 0.5 μg ml −1 Bevacizumab (histograms 2–5).

    Article Snippet: Reagents The pan VEGF antibody that recognizes all forms of VEGF-A was obtained from Santa Cruz (Dallas, TX, USA) (SC-507; used at 1:2,000).

    Techniques: In Vivo, Angiogenesis Assay, Electron Microscopy, Fluorescence, Staining, Isolation

    Hsp90 localizes to MVs and binds VEGF 90K . ( a ) Immunofluorescent (IF) images of primary tumour cells cultured from breast cancer patient-derived xenograft HCI-002 stained with antibodies against Hsp90 and VEGF. Arrows indicate Hsp90 and VEGF localized on MVs. Scale bar, 10 μm. ( b ) MVs from serum-starved MDAMB231 (lanes 1 and 3) or HeLa (lane 2) cells were isolated and lysed. Immunoprecipitations (IPs) using a pan VEGF antibody were performed on the MV lysates (25 μg MV protein, each; lanes 2 and 3). As a control, MV lysates were incubated without the VEGF antibody (lane 1). The immunocomplexes were blotted with VEGF and Hsp90 antibodies. MV lysates were probed with antibodies against Hsp90, VEGF, flotillin-2 and actin (to confirm equivalent amounts of sample were used in each IP) (bottom panel). ( c ) rVEGF 165 (30 ng) was incubated in RPMI medium with recombinant Hsp90 (rHsp90; 30 ng, lanes 2 and 3), and without (lane 2) or with recombinant tTG (100 ng, lane 3), for 1 h on ice to generate VEGF 90K . Immunoprecipitations (IPs) using an Hsp90 antibody were performed on the protein incubations and the immunocomplexes were blotted with antibodies against VEGF and Hsp90. As a control, rVEGF 165 was incubated without VEGF antibody (lane 1). ( d ) Lysates of MVs from MDAMB231 cells expressing control siRNA (lanes 1) or VEGF siRNA (lane 2) were immunoblotted with antibodies against VEGF, Hsp90 and the MV marker flotillin-2.

    Journal: Nature Communications

    Article Title: A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis

    doi: 10.1038/ncomms14450

    Figure Lengend Snippet: Hsp90 localizes to MVs and binds VEGF 90K . ( a ) Immunofluorescent (IF) images of primary tumour cells cultured from breast cancer patient-derived xenograft HCI-002 stained with antibodies against Hsp90 and VEGF. Arrows indicate Hsp90 and VEGF localized on MVs. Scale bar, 10 μm. ( b ) MVs from serum-starved MDAMB231 (lanes 1 and 3) or HeLa (lane 2) cells were isolated and lysed. Immunoprecipitations (IPs) using a pan VEGF antibody were performed on the MV lysates (25 μg MV protein, each; lanes 2 and 3). As a control, MV lysates were incubated without the VEGF antibody (lane 1). The immunocomplexes were blotted with VEGF and Hsp90 antibodies. MV lysates were probed with antibodies against Hsp90, VEGF, flotillin-2 and actin (to confirm equivalent amounts of sample were used in each IP) (bottom panel). ( c ) rVEGF 165 (30 ng) was incubated in RPMI medium with recombinant Hsp90 (rHsp90; 30 ng, lanes 2 and 3), and without (lane 2) or with recombinant tTG (100 ng, lane 3), for 1 h on ice to generate VEGF 90K . Immunoprecipitations (IPs) using an Hsp90 antibody were performed on the protein incubations and the immunocomplexes were blotted with antibodies against VEGF and Hsp90. As a control, rVEGF 165 was incubated without VEGF antibody (lane 1). ( d ) Lysates of MVs from MDAMB231 cells expressing control siRNA (lanes 1) or VEGF siRNA (lane 2) were immunoblotted with antibodies against VEGF, Hsp90 and the MV marker flotillin-2.

    Article Snippet: Reagents The pan VEGF antibody that recognizes all forms of VEGF-A was obtained from Santa Cruz (Dallas, TX, USA) (SC-507; used at 1:2,000).

    Techniques: Cell Culture, Derivative Assay, Staining, Isolation, Incubation, Recombinant, Expressing, Marker

    MV-associated VEGF 90K stimulates a sustained activation of VEGFRs that is insensitive to Bevacizumab. ( a ) Tubulogenesis assays were performed on HUVECs that were untreated (control; histogram 1) or treated with recombinant VEGF 165 (rVEGF; 15 ng ml −1 ) (histogram 2) or with MVs (10 μg ml −1 total protein) from MDAMB231 (histogram 3), HeLa (histogram 4) or SKBR3 (histogram 5) cells. ( b ) Lysates of serum-deprived HUVECs exposed to rVEGF 165 (5 ng ml −1 ; lanes 1–3) or MVs from MDAMB231 cells (5 μg ml −1 of MV protein; lanes 4–6) for the indicated lengths of time were immunoblotted with antibodies that recognize phosphorylated VEGFR2 (P-VEGFR2), total VEGFR2, phosphorylated ERK (P-ERK), total ERK or actin. ( c ) Lysates of serum-deprived HUVECs treated for the indicated time with MVs (5 μg ml −1 of MV protein) from MDAMB231 cells expressing VEGF siRNA were immunoblotted with antibodies that recognize phosphorylated VEGFR2, total VEGFR2 or actin. ( d ) Lysates of serum-deprived cultures of HUVECs that were untreated (lane 1), treated with MDAMB231 cell MVs (5 μg ml −1 of MV protein) without (lane 2) or with (lane 3) either 200 ng ml −1 anti-pan VEGF antibody, 0.5 μg ml −1 Bevacizumab (lane 4) or rVEGF 165 (5 ng ml −1 ) without (lane 5) or with (lane 6) 0.5 μg ml −1 Bevacizumab, for 15 min were immunoblotted with antibodies that recognize phosphorylated VEGFR2, total VEGFR2 or actin. ( e ) Lysates of serum-deprived HUVECs that were untreated (lane 1), or exposed to SKBR3 cell MVs (5 μg ml −1 total protein) treated without (lane 2) or with (lane 3) 0.5 μg ml −1 Bevacizumab for 15 min were immunoblotted with antibodies that recognize phosphorylated VEGFR2, total VEGFR2 or actin. ( f ) MDAMB231 cell MVs were evenly divided into two samples. In one sample, immunoprecipitations (IPs) using a pan VEGF antibody were performed on the intact MVs (∼25 μg of MV protein in RPMI medium; lane 1), while in the other sample, MVs were first lysed before immunoprecipitations were performed (lane 2). The immunocomplexes were blotted with a pan VEGF antibody and the MV protein inputs were blotted with an antibody against the MV marker flotillin-2.

    Journal: Nature Communications

    Article Title: A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis

    doi: 10.1038/ncomms14450

    Figure Lengend Snippet: MV-associated VEGF 90K stimulates a sustained activation of VEGFRs that is insensitive to Bevacizumab. ( a ) Tubulogenesis assays were performed on HUVECs that were untreated (control; histogram 1) or treated with recombinant VEGF 165 (rVEGF; 15 ng ml −1 ) (histogram 2) or with MVs (10 μg ml −1 total protein) from MDAMB231 (histogram 3), HeLa (histogram 4) or SKBR3 (histogram 5) cells. ( b ) Lysates of serum-deprived HUVECs exposed to rVEGF 165 (5 ng ml −1 ; lanes 1–3) or MVs from MDAMB231 cells (5 μg ml −1 of MV protein; lanes 4–6) for the indicated lengths of time were immunoblotted with antibodies that recognize phosphorylated VEGFR2 (P-VEGFR2), total VEGFR2, phosphorylated ERK (P-ERK), total ERK or actin. ( c ) Lysates of serum-deprived HUVECs treated for the indicated time with MVs (5 μg ml −1 of MV protein) from MDAMB231 cells expressing VEGF siRNA were immunoblotted with antibodies that recognize phosphorylated VEGFR2, total VEGFR2 or actin. ( d ) Lysates of serum-deprived cultures of HUVECs that were untreated (lane 1), treated with MDAMB231 cell MVs (5 μg ml −1 of MV protein) without (lane 2) or with (lane 3) either 200 ng ml −1 anti-pan VEGF antibody, 0.5 μg ml −1 Bevacizumab (lane 4) or rVEGF 165 (5 ng ml −1 ) without (lane 5) or with (lane 6) 0.5 μg ml −1 Bevacizumab, for 15 min were immunoblotted with antibodies that recognize phosphorylated VEGFR2, total VEGFR2 or actin. ( e ) Lysates of serum-deprived HUVECs that were untreated (lane 1), or exposed to SKBR3 cell MVs (5 μg ml −1 total protein) treated without (lane 2) or with (lane 3) 0.5 μg ml −1 Bevacizumab for 15 min were immunoblotted with antibodies that recognize phosphorylated VEGFR2, total VEGFR2 or actin. ( f ) MDAMB231 cell MVs were evenly divided into two samples. In one sample, immunoprecipitations (IPs) using a pan VEGF antibody were performed on the intact MVs (∼25 μg of MV protein in RPMI medium; lane 1), while in the other sample, MVs were first lysed before immunoprecipitations were performed (lane 2). The immunocomplexes were blotted with a pan VEGF antibody and the MV protein inputs were blotted with an antibody against the MV marker flotillin-2.

    Article Snippet: Reagents The pan VEGF antibody that recognizes all forms of VEGF-A was obtained from Santa Cruz (Dallas, TX, USA) (SC-507; used at 1:2,000).

    Techniques: Activation Assay, Recombinant, Expressing, Marker

    VEGF released from MVs regains its sensitivity to Bevacizumab. ( a ) Non-permeabilized MDAMB231 cells were analysed by immunofluorescent confocal microscopy using anti-VEGF and anti-Hsp90 antibodies. Top images: VEGF 90K and Hsp90 are detected on MVs (arrows). Bottom images: MDAMB231 cells treated with 10 μM 17AAG overnight were fixed and stained. Scale bar, 10 μm. Far-right: Blow-ups of the MVs. ( b ) MDAMB231 cell MVs treated without (lane 2) or with (lane 3) 17AAG at 37 °C for 2 h were lysed and immunoprecipitations were performed using a Hsp90 antibody (25 μg MV protein, each). ( c ) VEGF 90K was generated by tTG-catalysed crosslinking of rVEGF 165 , incubated with Hsp90 (30 ng), either without (lane 1) or with 17AAG (10 μM) (lane 2), at 37 °C for 1 h and immunoprecipitated using an anti-Hsp90 antibody. ( d ) MDAMB231 cell MVs treated without (lane 1) or with 17AAG (lane 2) at 37 °C for 2 h were collected on a 0.22 μm filter. The filtered MVs and the flow-through were immunoblotted. ( e ) Serum-deprived HUVECs were untreated (lane 1), or exposed to VEGF 90K (∼10 ng ml −1 ) released from 17AAG-treated MVs and present in the flow-through from the experiment shown in Fig. 5d , in the presence (lane 2) or absence (lane 3) of Bevacizumab, for 15 min, lysed and immunoblotted. ( f ) Serum-starved HUVECs, untreated (lanes 1, 2, 5 and 6) or pre-treated with 10 μM GA for 1 hour (lanes 3 and 4), were incubated without (lane 1) or with MVs (5 μg ml −1 protein) from MDAMB231 cells (lane 2), together with 200 ng ml −1 pan inactivating VEGF antibody (lane 4), 10 μM 17AAG (lane 5) or both the VEGF antibody and 17AAG (lane 6), for 15 min. Cell extracts were lysed and immunoblotted. ( g ) Serum-starved HUVECs were incubated with the VEGF 90K –Hsp90 complex (∼10 ng ml −1 ) without (lane 1) or with Bevacizumab (0.5 μg ml −1 ) (lane 2) for 15 min, lysed and immunoblotted. ( h ) Left: Relative amounts of tubulogenesis for HUVECs untreated (control; histogram 1) or treated with the VEGF 90K –Hsp90 complex (∼20 ng ml −1 total protein) without (histogram 3) or with 0.5 μg ml −1 Bevacizumab (histogram 4). Right: Images of the tubulogenesis assays.

    Journal: Nature Communications

    Article Title: A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis

    doi: 10.1038/ncomms14450

    Figure Lengend Snippet: VEGF released from MVs regains its sensitivity to Bevacizumab. ( a ) Non-permeabilized MDAMB231 cells were analysed by immunofluorescent confocal microscopy using anti-VEGF and anti-Hsp90 antibodies. Top images: VEGF 90K and Hsp90 are detected on MVs (arrows). Bottom images: MDAMB231 cells treated with 10 μM 17AAG overnight were fixed and stained. Scale bar, 10 μm. Far-right: Blow-ups of the MVs. ( b ) MDAMB231 cell MVs treated without (lane 2) or with (lane 3) 17AAG at 37 °C for 2 h were lysed and immunoprecipitations were performed using a Hsp90 antibody (25 μg MV protein, each). ( c ) VEGF 90K was generated by tTG-catalysed crosslinking of rVEGF 165 , incubated with Hsp90 (30 ng), either without (lane 1) or with 17AAG (10 μM) (lane 2), at 37 °C for 1 h and immunoprecipitated using an anti-Hsp90 antibody. ( d ) MDAMB231 cell MVs treated without (lane 1) or with 17AAG (lane 2) at 37 °C for 2 h were collected on a 0.22 μm filter. The filtered MVs and the flow-through were immunoblotted. ( e ) Serum-deprived HUVECs were untreated (lane 1), or exposed to VEGF 90K (∼10 ng ml −1 ) released from 17AAG-treated MVs and present in the flow-through from the experiment shown in Fig. 5d , in the presence (lane 2) or absence (lane 3) of Bevacizumab, for 15 min, lysed and immunoblotted. ( f ) Serum-starved HUVECs, untreated (lanes 1, 2, 5 and 6) or pre-treated with 10 μM GA for 1 hour (lanes 3 and 4), were incubated without (lane 1) or with MVs (5 μg ml −1 protein) from MDAMB231 cells (lane 2), together with 200 ng ml −1 pan inactivating VEGF antibody (lane 4), 10 μM 17AAG (lane 5) or both the VEGF antibody and 17AAG (lane 6), for 15 min. Cell extracts were lysed and immunoblotted. ( g ) Serum-starved HUVECs were incubated with the VEGF 90K –Hsp90 complex (∼10 ng ml −1 ) without (lane 1) or with Bevacizumab (0.5 μg ml −1 ) (lane 2) for 15 min, lysed and immunoblotted. ( h ) Left: Relative amounts of tubulogenesis for HUVECs untreated (control; histogram 1) or treated with the VEGF 90K –Hsp90 complex (∼20 ng ml −1 total protein) without (histogram 3) or with 0.5 μg ml −1 Bevacizumab (histogram 4). Right: Images of the tubulogenesis assays.

    Article Snippet: Reagents The pan VEGF antibody that recognizes all forms of VEGF-A was obtained from Santa Cruz (Dallas, TX, USA) (SC-507; used at 1:2,000).

    Techniques: Confocal Microscopy, Staining, Generated, Incubation, Immunoprecipitation, Flow Cytometry

    The role of MVs in Bevacizumab insensitivity extends to PDXs. ( a ) Lysates of serum-deprived HUVECs treated with conditioned medium (CM; 20 μg ml −1 total protein) from cell cultures of PDX samples HCI-001-003 and HCI-005 (Left) or HCI-006-011 and HCI-013 (Right), described in Table 1 , were supplemented without or with 0.5 μg ml −1 Bevacizumab, as indicated, for 15 min. HUVEC lysates were immunoblotted with antibodies that recognize phosphorylated VEGFR2, total VEGFR2 or actin. ( b ) MVs from cultures of cells established from PDX samples HCI-001 and HCI-002 were isolated, lysed and immunoblotted with antibodies against pan VEGF or the MV marker flotillin-2. ( c ) Serum-deprived HUVECs were incubated with serum-free medium alone (control; lane 1), or with serum-free medium containing the indicated combinations of MVs from PDX samples HCI-001 or HCI-002 (5 μg ml −1 of MV protein), 0.5 μg ml −1 Bevacizumab (lanes 3 and 6) and 10 μM 17AAG (lanes 4 and 7), for 15 min and then lysed. Cell extracts were immunoblotted with antibodies that recognize phosphorylated VEGFR2, total VEGFR2 or actin. ( d ) Plots showing relative mean tumour volumes (mm 3 ) of tumour graft HCI-003 in NOD/SCID mice that were untreated (vehicle control only), or treated with either Bevacizumab or Bevacizumab plus 17AAG. The differences between the tumour volumes for the Bevacizumab treatment group versus the vehicle only (control) group were statistically significant ( P

    Journal: Nature Communications

    Article Title: A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis

    doi: 10.1038/ncomms14450

    Figure Lengend Snippet: The role of MVs in Bevacizumab insensitivity extends to PDXs. ( a ) Lysates of serum-deprived HUVECs treated with conditioned medium (CM; 20 μg ml −1 total protein) from cell cultures of PDX samples HCI-001-003 and HCI-005 (Left) or HCI-006-011 and HCI-013 (Right), described in Table 1 , were supplemented without or with 0.5 μg ml −1 Bevacizumab, as indicated, for 15 min. HUVEC lysates were immunoblotted with antibodies that recognize phosphorylated VEGFR2, total VEGFR2 or actin. ( b ) MVs from cultures of cells established from PDX samples HCI-001 and HCI-002 were isolated, lysed and immunoblotted with antibodies against pan VEGF or the MV marker flotillin-2. ( c ) Serum-deprived HUVECs were incubated with serum-free medium alone (control; lane 1), or with serum-free medium containing the indicated combinations of MVs from PDX samples HCI-001 or HCI-002 (5 μg ml −1 of MV protein), 0.5 μg ml −1 Bevacizumab (lanes 3 and 6) and 10 μM 17AAG (lanes 4 and 7), for 15 min and then lysed. Cell extracts were immunoblotted with antibodies that recognize phosphorylated VEGFR2, total VEGFR2 or actin. ( d ) Plots showing relative mean tumour volumes (mm 3 ) of tumour graft HCI-003 in NOD/SCID mice that were untreated (vehicle control only), or treated with either Bevacizumab or Bevacizumab plus 17AAG. The differences between the tumour volumes for the Bevacizumab treatment group versus the vehicle only (control) group were statistically significant ( P

    Article Snippet: Reagents The pan VEGF antibody that recognizes all forms of VEGF-A was obtained from Santa Cruz (Dallas, TX, USA) (SC-507; used at 1:2,000).

    Techniques: Isolation, Marker, Incubation, Mouse Assay

    ( A ) Heart’s specimen from normal mice obtained 7 day after exposure to hypoxia. The expression of VEGF returns to initial levels. ( B ) Heart’s specimen from mdx mice obtained 7 day after exposure to hypoxia. The expression of VEGF in myocardium remains elevated but returns to normal in vessel walls. Scale bar =100 μm.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Cardiomyopathy in the mouse model of Duchenne muscular dystrophy caused by disordered secretion of vascular endothelial growth factor

    doi: 10.12659/MSM.882043

    Figure Lengend Snippet: ( A ) Heart’s specimen from normal mice obtained 7 day after exposure to hypoxia. The expression of VEGF returns to initial levels. ( B ) Heart’s specimen from mdx mice obtained 7 day after exposure to hypoxia. The expression of VEGF in myocardium remains elevated but returns to normal in vessel walls. Scale bar =100 μm.

    Article Snippet: The membranes were blocked in 0.05%Tween 20 for 1 h, then incubated with primary anti-VEGF-A (Santa Cruz Biotechnology) at 3 μl/ml in 0.05% Tween 20 in a humid chamber with orbital shaking for 1.5 h, followed by incubation with the secondary biotinylated antibody under similar conditions for 1 h. Proteins were visualized using the horseradish peroxidase-diaminobenzidine system (DAB Substrate Kit for Peroxidase; SK-4100, Vector Laboratories).

    Techniques: Mouse Assay, Expressing

    In situ hybridization analysis of VEGF mRNA expression in cardiac vessel endothelial cells ( A ) and in cardiac myocytes ( B ) from normal (C57BI/10ScSn) and mdx (C57BI/10ScSn) mice (values are optical densities expressed as percentage of the signal for control mice). N – 5 samples from each group, 5 fields of myocardium in 6 preparations from each mouse were examined together 150 analysis. * Statistically significant compared to normal – healthy mice.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Cardiomyopathy in the mouse model of Duchenne muscular dystrophy caused by disordered secretion of vascular endothelial growth factor

    doi: 10.12659/MSM.882043

    Figure Lengend Snippet: In situ hybridization analysis of VEGF mRNA expression in cardiac vessel endothelial cells ( A ) and in cardiac myocytes ( B ) from normal (C57BI/10ScSn) and mdx (C57BI/10ScSn) mice (values are optical densities expressed as percentage of the signal for control mice). N – 5 samples from each group, 5 fields of myocardium in 6 preparations from each mouse were examined together 150 analysis. * Statistically significant compared to normal – healthy mice.

    Article Snippet: The membranes were blocked in 0.05%Tween 20 for 1 h, then incubated with primary anti-VEGF-A (Santa Cruz Biotechnology) at 3 μl/ml in 0.05% Tween 20 in a humid chamber with orbital shaking for 1.5 h, followed by incubation with the secondary biotinylated antibody under similar conditions for 1 h. Proteins were visualized using the horseradish peroxidase-diaminobenzidine system (DAB Substrate Kit for Peroxidase; SK-4100, Vector Laboratories).

    Techniques: In Situ Hybridization, Expressing, Mouse Assay

    Immunohistochemical analysis of VEGF expression in cardiac vessel endothelial cells ( A ) and in cardiac myocytes ( B ) from normal (C57BI/10ScSn) and mdx (C57BI/10ScSn) mice (values are optical densities expressed as percentage of the signal for control mice).). N – 5 samples from each group, 5 fields of myocardium in 6 preparations from each mouse were examined together 150 analysis. * Statistically significant compared to normal – healthy mice.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Cardiomyopathy in the mouse model of Duchenne muscular dystrophy caused by disordered secretion of vascular endothelial growth factor

    doi: 10.12659/MSM.882043

    Figure Lengend Snippet: Immunohistochemical analysis of VEGF expression in cardiac vessel endothelial cells ( A ) and in cardiac myocytes ( B ) from normal (C57BI/10ScSn) and mdx (C57BI/10ScSn) mice (values are optical densities expressed as percentage of the signal for control mice).). N – 5 samples from each group, 5 fields of myocardium in 6 preparations from each mouse were examined together 150 analysis. * Statistically significant compared to normal – healthy mice.

    Article Snippet: The membranes were blocked in 0.05%Tween 20 for 1 h, then incubated with primary anti-VEGF-A (Santa Cruz Biotechnology) at 3 μl/ml in 0.05% Tween 20 in a humid chamber with orbital shaking for 1.5 h, followed by incubation with the secondary biotinylated antibody under similar conditions for 1 h. Proteins were visualized using the horseradish peroxidase-diaminobenzidine system (DAB Substrate Kit for Peroxidase; SK-4100, Vector Laboratories).

    Techniques: Immunohistochemistry, Expressing, Mouse Assay

    ( A ) Heart’s specimen from normal mice obtained 1 day after exposure to hypoxia. Increased expression of VEGF in both myocardium and vessel walls. ( B ) Heart’s specimen from mdx mice obtained 1 day after exposure to hypoxia. Low expression of VEGF in myocardium and an enhanced signal in vessel walls. Scale bar =100 μm.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Cardiomyopathy in the mouse model of Duchenne muscular dystrophy caused by disordered secretion of vascular endothelial growth factor

    doi: 10.12659/MSM.882043

    Figure Lengend Snippet: ( A ) Heart’s specimen from normal mice obtained 1 day after exposure to hypoxia. Increased expression of VEGF in both myocardium and vessel walls. ( B ) Heart’s specimen from mdx mice obtained 1 day after exposure to hypoxia. Low expression of VEGF in myocardium and an enhanced signal in vessel walls. Scale bar =100 μm.

    Article Snippet: The membranes were blocked in 0.05%Tween 20 for 1 h, then incubated with primary anti-VEGF-A (Santa Cruz Biotechnology) at 3 μl/ml in 0.05% Tween 20 in a humid chamber with orbital shaking for 1.5 h, followed by incubation with the secondary biotinylated antibody under similar conditions for 1 h. Proteins were visualized using the horseradish peroxidase-diaminobenzidine system (DAB Substrate Kit for Peroxidase; SK-4100, Vector Laboratories).

    Techniques: Mouse Assay, Expressing

    ( A ) Western analysis of VEGF expression in the heart following hypoxia normal mice. Control group (1); immediately after (2); 1 d ay after (3); 3 days after (4); 7 days after (5), and 21 days after (6) hypoxia. ( B ) Western analysis of VEGF expression in the heart following hypobaric hypoxia in dystrophic mice. Control group (1); immediately after (2); 1 day after (3); 3 days after (4); 7 days after (5), and 21 days after (6) hypoxia. ( C ) Quantitative analysis of western blot signals in normal and mdx mice. Note the difference in the timing of maximum VEGF expression between normal and mdx mice. The control group signal is set to 100%. * Statistically significant compared to healthy mice. In each group was 10 mice.

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Cardiomyopathy in the mouse model of Duchenne muscular dystrophy caused by disordered secretion of vascular endothelial growth factor

    doi: 10.12659/MSM.882043

    Figure Lengend Snippet: ( A ) Western analysis of VEGF expression in the heart following hypoxia normal mice. Control group (1); immediately after (2); 1 d ay after (3); 3 days after (4); 7 days after (5), and 21 days after (6) hypoxia. ( B ) Western analysis of VEGF expression in the heart following hypobaric hypoxia in dystrophic mice. Control group (1); immediately after (2); 1 day after (3); 3 days after (4); 7 days after (5), and 21 days after (6) hypoxia. ( C ) Quantitative analysis of western blot signals in normal and mdx mice. Note the difference in the timing of maximum VEGF expression between normal and mdx mice. The control group signal is set to 100%. * Statistically significant compared to healthy mice. In each group was 10 mice.

    Article Snippet: The membranes were blocked in 0.05%Tween 20 for 1 h, then incubated with primary anti-VEGF-A (Santa Cruz Biotechnology) at 3 μl/ml in 0.05% Tween 20 in a humid chamber with orbital shaking for 1.5 h, followed by incubation with the secondary biotinylated antibody under similar conditions for 1 h. Proteins were visualized using the horseradish peroxidase-diaminobenzidine system (DAB Substrate Kit for Peroxidase; SK-4100, Vector Laboratories).

    Techniques: Western Blot, Expressing, Mouse Assay

    FLI1 and PKC co-activation mediated hESCs differentiation into iECs a Schematic illustration of the EC differentiation strategy from hESCs. b Typical morphological images of hESC-EC differentiation on days of 0, 1, 2, and 3. Scale bar, 100 μm. c The ratio of CD31+/CD144+ cells gradually increased during induction. Columns represent the mean ± SD; n = 5 independent differentiation experiments. d Representative results of the percentage of CD31+/CD144+ cells during the induction process detected by FCM. e Overexpression of FLI1 and activation of PKC in different hESC lines (hESC-254 or hESC-137) induced iECs. f Overexpressing FLI1 with different PKC activators (PMA or prostratin) yielded iECs. g Expression levels of VEGF , GATA2 , CD31 and CD144 genes in hESCs, human fibroblasts (HFs), iECs and EPCs. Columns represent the mean ± SD; n = 5 independent differentiation experiments

    Journal: Cell Death & Disease

    Article Title: FLI1 and PKC co-activation promote highly efficient differentiation of human embryonic stem cells into endothelial-like cells

    doi: 10.1038/s41419-017-0162-9

    Figure Lengend Snippet: FLI1 and PKC co-activation mediated hESCs differentiation into iECs a Schematic illustration of the EC differentiation strategy from hESCs. b Typical morphological images of hESC-EC differentiation on days of 0, 1, 2, and 3. Scale bar, 100 μm. c The ratio of CD31+/CD144+ cells gradually increased during induction. Columns represent the mean ± SD; n = 5 independent differentiation experiments. d Representative results of the percentage of CD31+/CD144+ cells during the induction process detected by FCM. e Overexpression of FLI1 and activation of PKC in different hESC lines (hESC-254 or hESC-137) induced iECs. f Overexpressing FLI1 with different PKC activators (PMA or prostratin) yielded iECs. g Expression levels of VEGF , GATA2 , CD31 and CD144 genes in hESCs, human fibroblasts (HFs), iECs and EPCs. Columns represent the mean ± SD; n = 5 independent differentiation experiments

    Article Snippet: After 3 days, supplements were changed with 50 ng ml−1 VEGF-A (96-100-20-10, PEPROTECH).

    Techniques: Activation Assay, Over Expression, Expressing

    Assessment of VEGF-A-induced cell proliferation and migration upon modulation of miR-424–5p or miR-29a-3p. ( A ) Cell proliferation assessed by cytofluorimetric analysis of EdU incorporation into the DNA in control spheroids (SPHC) or VEGF-A-stimulated spheroids (SPHV) generated with ECs transfected with miR-424–5p or miR-29a-3p mimics or inhibitors or their respective controls. Representative plots for n = 3 experiments. ( B ) Cell proliferation assessed by cytofluorimetric analysis of EdU incorporation into the DNA during cell replication in 2D cultured HUVECs transfected with miR-424–5p or miR-29a-3p mimics or inhibitors, or respective transfection controls. The assay was performed after 18 hr of VEGF stimulation. Data are represented as mean ± SEM from n = 3 experiments. ( C ) VEGF-induced cell migration of HUVECs transfected with miR-424–5p or miR-29a-3p mimics or inhibitors, or respective transfection controls, assessed by xCELLigence and represented as the slope of the migration curve over a 24 hr experiment. Data are represented as mean ± SEM from n = 3 experiments. ***, p

    Journal: eLife

    Article Title: A regulatory microRNA network controls endothelial cell phenotypic switch during sprouting angiogenesis

    doi: 10.7554/eLife.48095

    Figure Lengend Snippet: Assessment of VEGF-A-induced cell proliferation and migration upon modulation of miR-424–5p or miR-29a-3p. ( A ) Cell proliferation assessed by cytofluorimetric analysis of EdU incorporation into the DNA in control spheroids (SPHC) or VEGF-A-stimulated spheroids (SPHV) generated with ECs transfected with miR-424–5p or miR-29a-3p mimics or inhibitors or their respective controls. Representative plots for n = 3 experiments. ( B ) Cell proliferation assessed by cytofluorimetric analysis of EdU incorporation into the DNA during cell replication in 2D cultured HUVECs transfected with miR-424–5p or miR-29a-3p mimics or inhibitors, or respective transfection controls. The assay was performed after 18 hr of VEGF stimulation. Data are represented as mean ± SEM from n = 3 experiments. ( C ) VEGF-induced cell migration of HUVECs transfected with miR-424–5p or miR-29a-3p mimics or inhibitors, or respective transfection controls, assessed by xCELLigence and represented as the slope of the migration curve over a 24 hr experiment. Data are represented as mean ± SEM from n = 3 experiments. ***, p

    Article Snippet: Sprouting was induced by addition of 20 ng/ml recombinant human VEGF-A (R and D Systems, Minneapolis, MN, USA) to the collagen solution and to the overlaying medium.

    Techniques: Migration, Generated, Transfection, Cell Culture

    DICER knock-down. ( A ) Sprouting assay performed with spheroids generated with cells that had been transduced with a non-targeting shRNA ( DICER WT shCtr) or with two different shRNAs targeting DICER ( DICER KD sh#3 and DICER KD sh#4), and the corresponding quantification of the sprouted area. Data are represented as mean ± SEM from n = 6 experiments. Scale bars, 200 µm. ( B ) DICER expression measured by real-time PCR assay in cells transduced with a non-targeting shRNA ( DICER WT shCtr) or with two different shRNAs targeting DICER ( DICER KD sh#3 and DICER KD sh#4). Data are represented as mean ± SEM from n = 3 experiments. ( C ) GSEA study performed against the canonical pathways gene sets collection, comparing control spheroids (SPHC) generated with DICER KD or DICER WT cells. FDR q value > 0.05, not significant. ( D ) VEGF-A-induced cell proliferation assessed by cytofluorimetric analysis of EdU incorporation into the DNA during cell replication, in 2D-cultured HUVECs. Cells were transduced either with a scrambled shRNA ( DICER WT ) or with a shRNA targeting DICER ( DICER KD ). Plots are representative of n = 3 experiments. Data represent mean percentage of proliferating cells ± SEM from n = 3 experiments. p

    Journal: eLife

    Article Title: A regulatory microRNA network controls endothelial cell phenotypic switch during sprouting angiogenesis

    doi: 10.7554/eLife.48095

    Figure Lengend Snippet: DICER knock-down. ( A ) Sprouting assay performed with spheroids generated with cells that had been transduced with a non-targeting shRNA ( DICER WT shCtr) or with two different shRNAs targeting DICER ( DICER KD sh#3 and DICER KD sh#4), and the corresponding quantification of the sprouted area. Data are represented as mean ± SEM from n = 6 experiments. Scale bars, 200 µm. ( B ) DICER expression measured by real-time PCR assay in cells transduced with a non-targeting shRNA ( DICER WT shCtr) or with two different shRNAs targeting DICER ( DICER KD sh#3 and DICER KD sh#4). Data are represented as mean ± SEM from n = 3 experiments. ( C ) GSEA study performed against the canonical pathways gene sets collection, comparing control spheroids (SPHC) generated with DICER KD or DICER WT cells. FDR q value > 0.05, not significant. ( D ) VEGF-A-induced cell proliferation assessed by cytofluorimetric analysis of EdU incorporation into the DNA during cell replication, in 2D-cultured HUVECs. Cells were transduced either with a scrambled shRNA ( DICER WT ) or with a shRNA targeting DICER ( DICER KD ). Plots are representative of n = 3 experiments. Data represent mean percentage of proliferating cells ± SEM from n = 3 experiments. p

    Article Snippet: Sprouting was induced by addition of 20 ng/ml recombinant human VEGF-A (R and D Systems, Minneapolis, MN, USA) to the collagen solution and to the overlaying medium.

    Techniques: Generated, Transduction, shRNA, Expressing, Real-time Polymerase Chain Reaction, Cell Culture

    Assessment of the effective concentrations of ERK and p38 inhibitors in ECs. ( A ) Western blot showing ERK phosphorylation, detected with an anti-phospho-ERK antibody (P-ERK), upon VEGF-A stimulus in the presence of an increasing concentration (10, 100, 300 nM) of the ERK inhibitor SHC 772984, or vehicle (DMSO). Total ERK was used as protein loading control. ( B ) Western blot showing p38 phosphorylation, detected with an anti-phospho-p38 antibody (P–p38), upon VEGF-A stimulus in the presence of increasing concentration (10, 100, 300 μM) of the p38 inhibitor SB 202190, or vehicle (DMSO). Total p38 was used as protein loading control. Data are representative of three independent experiments.

    Journal: eLife

    Article Title: A regulatory microRNA network controls endothelial cell phenotypic switch during sprouting angiogenesis

    doi: 10.7554/eLife.48095

    Figure Lengend Snippet: Assessment of the effective concentrations of ERK and p38 inhibitors in ECs. ( A ) Western blot showing ERK phosphorylation, detected with an anti-phospho-ERK antibody (P-ERK), upon VEGF-A stimulus in the presence of an increasing concentration (10, 100, 300 nM) of the ERK inhibitor SHC 772984, or vehicle (DMSO). Total ERK was used as protein loading control. ( B ) Western blot showing p38 phosphorylation, detected with an anti-phospho-p38 antibody (P–p38), upon VEGF-A stimulus in the presence of increasing concentration (10, 100, 300 μM) of the p38 inhibitor SB 202190, or vehicle (DMSO). Total p38 was used as protein loading control. Data are representative of three independent experiments.

    Article Snippet: Sprouting was induced by addition of 20 ng/ml recombinant human VEGF-A (R and D Systems, Minneapolis, MN, USA) to the collagen solution and to the overlaying medium.

    Techniques: Western Blot, Concentration Assay

    Cellular proliferation, matrix metalloproteinase 2 ( MMP-2 ), and matrix metalloproteinase 9 ( MMP-9 ) are decreased in lentivirus (LV)–small hairpin RNA (shRNA)–vascular endothelial growth factor-A ( VEGF-A )–transduced vessels. ( a , upper panel) Representative sections after Ki-67 staining at the venous stenosis of the LV-shRNA- VEGF-A (LV) and scrambled shRNA (control (C)) or Avastin-treated vessels or immunoglobulin G (IgG) controls at days 14 (D14) and 28 (D28). Nuclei staining brown are positive for Ki-67. IgG antibody staining was performed to serve as negative controls. *Indicates the lumen. All are original magnification × 40. Bar=200 μm. Pooled data for the LV and C groups or Avastin-treated vessels or IgG controls are shown in the lower panel. This demonstrates a significant decrease in the mean Ki-67 index in the LV-transduced vessels when compared with the C vessels at day 14 ( P

    Journal: Kidney International

    Article Title: Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation

    doi: 10.1038/ki.2013.290

    Figure Lengend Snippet: Cellular proliferation, matrix metalloproteinase 2 ( MMP-2 ), and matrix metalloproteinase 9 ( MMP-9 ) are decreased in lentivirus (LV)–small hairpin RNA (shRNA)–vascular endothelial growth factor-A ( VEGF-A )–transduced vessels. ( a , upper panel) Representative sections after Ki-67 staining at the venous stenosis of the LV-shRNA- VEGF-A (LV) and scrambled shRNA (control (C)) or Avastin-treated vessels or immunoglobulin G (IgG) controls at days 14 (D14) and 28 (D28). Nuclei staining brown are positive for Ki-67. IgG antibody staining was performed to serve as negative controls. *Indicates the lumen. All are original magnification × 40. Bar=200 μm. Pooled data for the LV and C groups or Avastin-treated vessels or IgG controls are shown in the lower panel. This demonstrates a significant decrease in the mean Ki-67 index in the LV-transduced vessels when compared with the C vessels at day 14 ( P

    Article Snippet: We determined whether Avastin, a humanized monoclonal antibody to VEGF-A (Bevacizumab, Genentech, San Francisco, CA), would decrease VNH formation.

    Techniques: shRNA, Staining

    Smooth muscle cell index and vascular endothelial growth factor receptor 1 ( VEGFR-1 ) expression are reduced in lentivirus (LV)–small hairpin RNA (shRNA)–vascular endothelial growth factor-A ( VEGF-A )–transduced vessels. ( a , upper panel) Representative sections after α-smooth muscle actin (α-SMA) staining at the venous stenosis of the LV-shRNA- VEGF-A (LV) and scrambled shRNA (control (C)) and Avastin treated with control vessels at days 14 and 28. Cells staining brown are positive for α-SMA. immunoglobulin G (IgG) antibody staining was performed to serve as negative control. *Indicates the lumen. All are original magnification × 40. Bar=200 μm. Pooled data for the LV and C groups and Avastin-treated and control vessels are shown in a (lower panel). This demonstrates a significant reduction in the average α-SMA index in LV-transduced vessels when compared with C vessels by day 21 ( P

    Journal: Kidney International

    Article Title: Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation

    doi: 10.1038/ki.2013.290

    Figure Lengend Snippet: Smooth muscle cell index and vascular endothelial growth factor receptor 1 ( VEGFR-1 ) expression are reduced in lentivirus (LV)–small hairpin RNA (shRNA)–vascular endothelial growth factor-A ( VEGF-A )–transduced vessels. ( a , upper panel) Representative sections after α-smooth muscle actin (α-SMA) staining at the venous stenosis of the LV-shRNA- VEGF-A (LV) and scrambled shRNA (control (C)) and Avastin treated with control vessels at days 14 and 28. Cells staining brown are positive for α-SMA. immunoglobulin G (IgG) antibody staining was performed to serve as negative control. *Indicates the lumen. All are original magnification × 40. Bar=200 μm. Pooled data for the LV and C groups and Avastin-treated and control vessels are shown in a (lower panel). This demonstrates a significant reduction in the average α-SMA index in LV-transduced vessels when compared with C vessels by day 21 ( P

    Article Snippet: We determined whether Avastin, a humanized monoclonal antibody to VEGF-A (Bevacizumab, Genentech, San Francisco, CA), would decrease VNH formation.

    Techniques: Expressing, shRNA, Staining, Negative Control

    Hematoxylin and eosin (H E) and picrosirius red staining of the lentivirus (LV)–small hairpin RNA (shRNA)–vascular endothelial growth factor-A ( VEGF-A )–transduced and Avastin-treated vessels have increased lumen vessel area with decreased media and adventitia area and collagen expression. ( a , first column) Representative sections after H E at the venous stenosis of the LV-shRNA- VEGF-A (LV) and scrambled- VEGF-A (control (C))–transduced vessels or Avastin-treated vessels or immunoglobulin G (IgG) controls at day 14 showing increase in lumen vessel area. ( a , second column) Representative polarized light microscopy of picrosirius red–stained venous stenosis showing decreased fibrosis (collagen fibers are bright yellow) of the LV and C-transduced vessels and Avastin-treated vessels and controls. Qualitatively, there is a reduction in collagen staining by Sirius red by days 3–21. *Indicates the lumen. Bar=200 μm. Pooled data for mean lumen vessel area LV and C groups and Avastin-treated and control vessels are shown in b . There is a significant increase in the mean lumen vessel area in the LV-transduced vessels when compared with C vessels for days 14–28 ( P

    Journal: Kidney International

    Article Title: Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation

    doi: 10.1038/ki.2013.290

    Figure Lengend Snippet: Hematoxylin and eosin (H E) and picrosirius red staining of the lentivirus (LV)–small hairpin RNA (shRNA)–vascular endothelial growth factor-A ( VEGF-A )–transduced and Avastin-treated vessels have increased lumen vessel area with decreased media and adventitia area and collagen expression. ( a , first column) Representative sections after H E at the venous stenosis of the LV-shRNA- VEGF-A (LV) and scrambled- VEGF-A (control (C))–transduced vessels or Avastin-treated vessels or immunoglobulin G (IgG) controls at day 14 showing increase in lumen vessel area. ( a , second column) Representative polarized light microscopy of picrosirius red–stained venous stenosis showing decreased fibrosis (collagen fibers are bright yellow) of the LV and C-transduced vessels and Avastin-treated vessels and controls. Qualitatively, there is a reduction in collagen staining by Sirius red by days 3–21. *Indicates the lumen. Bar=200 μm. Pooled data for mean lumen vessel area LV and C groups and Avastin-treated and control vessels are shown in b . There is a significant increase in the mean lumen vessel area in the LV-transduced vessels when compared with C vessels for days 14–28 ( P

    Article Snippet: We determined whether Avastin, a humanized monoclonal antibody to VEGF-A (Bevacizumab, Genentech, San Francisco, CA), would decrease VNH formation.

    Techniques: Staining, shRNA, Expressing, Light Microscopy

    There is decreased proliferation, invasion, α-smooth muscle actin (α-SMA), and matrix metalloproteinase 2 ( MMP-2 ) expression with increased caspase 3 activities in the lentivirus (LV)–small hairpin RNA (shRNA)–vascular endothelial growth factor-A ( VEGF-A )–transduced cells subjected to hypoxia. Western blot for α-SMA after transduction LV-shRNA- VEGF-A (LV) and scrambled shRNA- VEGF-A (C) in AKR-2B fibroblasts subjected to hypoxia at 24 and 72 h. A typical western blot is shown in the upper panel and the pooled data in the lower panel ( a ). This demonstrates a significant reduction in the mean α-SMA expression in the LV-transduced cells when compared with C cells at 24 ( P

    Journal: Kidney International

    Article Title: Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation

    doi: 10.1038/ki.2013.290

    Figure Lengend Snippet: There is decreased proliferation, invasion, α-smooth muscle actin (α-SMA), and matrix metalloproteinase 2 ( MMP-2 ) expression with increased caspase 3 activities in the lentivirus (LV)–small hairpin RNA (shRNA)–vascular endothelial growth factor-A ( VEGF-A )–transduced cells subjected to hypoxia. Western blot for α-SMA after transduction LV-shRNA- VEGF-A (LV) and scrambled shRNA- VEGF-A (C) in AKR-2B fibroblasts subjected to hypoxia at 24 and 72 h. A typical western blot is shown in the upper panel and the pooled data in the lower panel ( a ). This demonstrates a significant reduction in the mean α-SMA expression in the LV-transduced cells when compared with C cells at 24 ( P

    Article Snippet: We determined whether Avastin, a humanized monoclonal antibody to VEGF-A (Bevacizumab, Genentech, San Francisco, CA), would decrease VNH formation.

    Techniques: Expressing, shRNA, Western Blot, Transduction

    Cartoon of proposed mechanism. Schematic showing ( a ) normal vein, ( b ) vein after arteriovenous fistula (AVF) placement, and ( c ) outflow vein after fistula placement with lentivirus (LV)–small hairpin RNA (shRNA)–vascular endothelial growth factor-A ( VEGF-A ) silencing and its different mechanisms. HIF-1α, hypoxia-inducible factor-1α.

    Journal: Kidney International

    Article Title: Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation

    doi: 10.1038/ki.2013.290

    Figure Lengend Snippet: Cartoon of proposed mechanism. Schematic showing ( a ) normal vein, ( b ) vein after arteriovenous fistula (AVF) placement, and ( c ) outflow vein after fistula placement with lentivirus (LV)–small hairpin RNA (shRNA)–vascular endothelial growth factor-A ( VEGF-A ) silencing and its different mechanisms. HIF-1α, hypoxia-inducible factor-1α.

    Article Snippet: We determined whether Avastin, a humanized monoclonal antibody to VEGF-A (Bevacizumab, Genentech, San Francisco, CA), would decrease VNH formation.

    Techniques: shRNA

    ( a ) Representative sections from TdT-mediated dNTP nick end labeling (TUNEL) staining at the venous stenosis of the LV-shRNA- VEGF-A (LV) with scrambled- VEGF-A (control (C))–transduced control vessels or Avastin-treated with immunoglobulin G (IgG) controls at days 14 and 28. Nuclei staining brown are positive for TUNEL. Negative control is shown where the recombinant terminal deoxynucleotidyl transferase enzyme was omitted. All are original magnification × 40. Bar=200 μm. *Indicates the lumen. Pooled data for LV- and C-transduced vessels or Avastin-treated or IgG controls are shown in b . This demonstrates a significant increase in the mean TUNEL index at day 14 ( P

    Journal: Kidney International

    Article Title: Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation

    doi: 10.1038/ki.2013.290

    Figure Lengend Snippet: ( a ) Representative sections from TdT-mediated dNTP nick end labeling (TUNEL) staining at the venous stenosis of the LV-shRNA- VEGF-A (LV) with scrambled- VEGF-A (control (C))–transduced control vessels or Avastin-treated with immunoglobulin G (IgG) controls at days 14 and 28. Nuclei staining brown are positive for TUNEL. Negative control is shown where the recombinant terminal deoxynucleotidyl transferase enzyme was omitted. All are original magnification × 40. Bar=200 μm. *Indicates the lumen. Pooled data for LV- and C-transduced vessels or Avastin-treated or IgG controls are shown in b . This demonstrates a significant increase in the mean TUNEL index at day 14 ( P

    Article Snippet: We determined whether Avastin, a humanized monoclonal antibody to VEGF-A (Bevacizumab, Genentech, San Francisco, CA), would decrease VNH formation.

    Techniques: End Labeling, TUNEL Assay, Staining, shRNA, Negative Control, Recombinant

    Vascular endothelial growth factor-A ( VEGF-A ) expression is reduced in lentivirus (LV)–small hairpin RNA (shRNA)- VEGF-A -transduced and Avastin-treated vessels with decreased CD31 staining. ( a , first to third columns) In situ hybridization of mRNA for VEGF-A in the LV-transduced vessels when compared with scrambled-shRNA- VEGF-A (control (C)) vessels, with arrows on cells positive for VEGF-A mRNA expression (brown). By day 3 (D3), there was a reduction of mRNA for VEGF-A being localized to the media and adventitia and by day 7 (D7), it was localized to the media and intima. In contrast, the vessels transduced with C shRNA showed increased mRNA expression of VEGF-A in the adventitia and media by day 3, and in the media and intima by day 7. ( b ) Pooled data for the in situ transcript levels of VEGF-A in the outflow vein of the LV-transduced vessels that was significantly reduced when compared with C vessels at day 7 ( P

    Journal: Kidney International

    Article Title: Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation

    doi: 10.1038/ki.2013.290

    Figure Lengend Snippet: Vascular endothelial growth factor-A ( VEGF-A ) expression is reduced in lentivirus (LV)–small hairpin RNA (shRNA)- VEGF-A -transduced and Avastin-treated vessels with decreased CD31 staining. ( a , first to third columns) In situ hybridization of mRNA for VEGF-A in the LV-transduced vessels when compared with scrambled-shRNA- VEGF-A (control (C)) vessels, with arrows on cells positive for VEGF-A mRNA expression (brown). By day 3 (D3), there was a reduction of mRNA for VEGF-A being localized to the media and adventitia and by day 7 (D7), it was localized to the media and intima. In contrast, the vessels transduced with C shRNA showed increased mRNA expression of VEGF-A in the adventitia and media by day 3, and in the media and intima by day 7. ( b ) Pooled data for the in situ transcript levels of VEGF-A in the outflow vein of the LV-transduced vessels that was significantly reduced when compared with C vessels at day 7 ( P

    Article Snippet: We determined whether Avastin, a humanized monoclonal antibody to VEGF-A (Bevacizumab, Genentech, San Francisco, CA), would decrease VNH formation.

    Techniques: Expressing, shRNA, Staining, In Situ Hybridization, Transduction, In Situ

    There is decreased hypoxia-inducible factor-1α (HIF-1α) expression and staining in lentivirus (LV)–small hairpin RNA (shRNA)–vascular endothelial growth factor-A ( VEGF-A )–transduced and Avastin-treated vessels. ( a ) Real-time PCR (RT- PCR) analysis for HIF-1α expression after transduction with LV and control shRNA (C). A typical blot is shown in the upper panel and the pooled data in the lower panel. This demonstrates a significant reduction in average HIF-1α expression in the LV-transduced vessels when compared with C vessels at days 7 (D7; P

    Journal: Kidney International

    Article Title: Adventitial transduction of lentivirus-shRNA-VEGF-A in arteriovenous fistula reduces venous stenosis formation

    doi: 10.1038/ki.2013.290

    Figure Lengend Snippet: There is decreased hypoxia-inducible factor-1α (HIF-1α) expression and staining in lentivirus (LV)–small hairpin RNA (shRNA)–vascular endothelial growth factor-A ( VEGF-A )–transduced and Avastin-treated vessels. ( a ) Real-time PCR (RT- PCR) analysis for HIF-1α expression after transduction with LV and control shRNA (C). A typical blot is shown in the upper panel and the pooled data in the lower panel. This demonstrates a significant reduction in average HIF-1α expression in the LV-transduced vessels when compared with C vessels at days 7 (D7; P

    Article Snippet: We determined whether Avastin, a humanized monoclonal antibody to VEGF-A (Bevacizumab, Genentech, San Francisco, CA), would decrease VNH formation.

    Techniques: Expressing, Staining, shRNA, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transduction

    Pleiotropic effects of NGF on HUVEC . (A) Growth assay. HUVEC cultured on standard culture plastic were treated with NGF (100 ng/ml), or VEGF (10 ng/ml) for 48 h. (B and C) Migration and invasion assay using Transwells. (D) Cord formation assay on matrigel-coated 24-well dishes. (E) Endothelial cell monolayer permeability assay. The kinetics of 70 kDa Dextran-FITC having passed through the monolayer of endothelial cells was determined by measurement of fluorescence in the lower chambers at different time points. A to D Student's t-test, *p

    Journal: Molecular Cancer

    Article Title: Nerve growth factor promotes breast cancer angiogenesis by activating multiple pathways

    doi: 10.1186/1476-4598-9-157

    Figure Lengend Snippet: Pleiotropic effects of NGF on HUVEC . (A) Growth assay. HUVEC cultured on standard culture plastic were treated with NGF (100 ng/ml), or VEGF (10 ng/ml) for 48 h. (B and C) Migration and invasion assay using Transwells. (D) Cord formation assay on matrigel-coated 24-well dishes. (E) Endothelial cell monolayer permeability assay. The kinetics of 70 kDa Dextran-FITC having passed through the monolayer of endothelial cells was determined by measurement of fluorescence in the lower chambers at different time points. A to D Student's t-test, *p

    Article Snippet: Reagents Human recombinant NGF and VEGF, neutralizing antibodies against NGF, VEGF and the corresponding isotype control antibodies were purchased from R & D Systems.

    Techniques: Growth Assay, Cell Culture, Migration, Invasion Assay, Tube Formation Assay, Permeability, Fluorescence

    Involvement of the VEGF in NGF-stimulated angiogenesis . (A) ELISA quantification of VEGF in conditioned media from HUVEC and MDA-MB-231 cells. Cells were treated with NGF (100 ng/ml) for 6 h or 24 h, conditioned media were concentrated before ELISA assay, as described in materials and methods. (B) Invasion assay using Transwells. HUVEC were treated with NGF (100 ng/ml) or VEGF (10 ng/ml) in the presence of isotype control or anti-VEGF neutralizing antibodies (1 μg/ml) for 24 h. (C) In vivo angiogenesis assay. Matrigel containing a mixture of NGF and isotype control or anti-VEGF neutralizing antibodies (37.5 μg/ml) was subcutaneously injected into SCID mice (five mice per group) as described in materials and methods. Hemoglobin in Matrigel plugs was quantified by Drabkin method 7 days after injection. Results are the mean of three independent experiments. Student's t-test, *p

    Journal: Molecular Cancer

    Article Title: Nerve growth factor promotes breast cancer angiogenesis by activating multiple pathways

    doi: 10.1186/1476-4598-9-157

    Figure Lengend Snippet: Involvement of the VEGF in NGF-stimulated angiogenesis . (A) ELISA quantification of VEGF in conditioned media from HUVEC and MDA-MB-231 cells. Cells were treated with NGF (100 ng/ml) for 6 h or 24 h, conditioned media were concentrated before ELISA assay, as described in materials and methods. (B) Invasion assay using Transwells. HUVEC were treated with NGF (100 ng/ml) or VEGF (10 ng/ml) in the presence of isotype control or anti-VEGF neutralizing antibodies (1 μg/ml) for 24 h. (C) In vivo angiogenesis assay. Matrigel containing a mixture of NGF and isotype control or anti-VEGF neutralizing antibodies (37.5 μg/ml) was subcutaneously injected into SCID mice (five mice per group) as described in materials and methods. Hemoglobin in Matrigel plugs was quantified by Drabkin method 7 days after injection. Results are the mean of three independent experiments. Student's t-test, *p

    Article Snippet: Reagents Human recombinant NGF and VEGF, neutralizing antibodies against NGF, VEGF and the corresponding isotype control antibodies were purchased from R & D Systems.

    Techniques: Enzyme-linked Immunosorbent Assay, Multiple Displacement Amplification, Invasion Assay, In Vivo, Angiogenesis Assay, Injection, Mouse Assay

    Angiogenesis assay using Matrigel plugs in SCID mice . Matrigel containing different reagents was subcutaneously injected into SCID mice as described in materials and methods. (A, B) Matrigel was mixed with MDA-MB-231 breast cancer cells and isotype control or anti-NGF neutralizing antibodies (75 μg/ml); (C) Matrigel was mixed with proNGF (7.5 μg/ml), NGF (3.75 μg/ml), or VEGF (0.375 μg/ml). Angiogenesis was analyzed by quantification of hemoglobin (A, C) and microvessel density (B) as described in materials and methods. Five mice were used for each group and results are the mean of three independent experiments. Student's t-test, *p

    Journal: Molecular Cancer

    Article Title: Nerve growth factor promotes breast cancer angiogenesis by activating multiple pathways

    doi: 10.1186/1476-4598-9-157

    Figure Lengend Snippet: Angiogenesis assay using Matrigel plugs in SCID mice . Matrigel containing different reagents was subcutaneously injected into SCID mice as described in materials and methods. (A, B) Matrigel was mixed with MDA-MB-231 breast cancer cells and isotype control or anti-NGF neutralizing antibodies (75 μg/ml); (C) Matrigel was mixed with proNGF (7.5 μg/ml), NGF (3.75 μg/ml), or VEGF (0.375 μg/ml). Angiogenesis was analyzed by quantification of hemoglobin (A, C) and microvessel density (B) as described in materials and methods. Five mice were used for each group and results are the mean of three independent experiments. Student's t-test, *p

    Article Snippet: Reagents Human recombinant NGF and VEGF, neutralizing antibodies against NGF, VEGF and the corresponding isotype control antibodies were purchased from R & D Systems.

    Techniques: Angiogenesis Assay, Mouse Assay, Injection, Multiple Displacement Amplification

    fVII is internalized by TF-expressing VEGF-stimulated angiogenic HUVECs and MDA-MB-231 breast cancer cells. fVII protein primarily bound to the cell plasma membrane ( green, arrows in a and d ) of the fixed cells (0 min), while endocytosis of fVII, shown as co-localization intracellularly with Alexa Fluor 633-phalloidin-stained cytoskeletal F-actin ( red, arrows in b and e ), was observed at 37°C for 30 min after incubation with VEGF-stimulated HUVECs ( b ) and human breast cancer MDA-MB-231 cells ( e ). c and f Secondary antibody FITC controls for non-specific FITC background. Scale bars , 20 µm

    Journal: Breast cancer research and treatment

    Article Title: Selective and effective killing of angiogenic vascular endothelial cells and cancer cells by targeting tissue factor using a factor VII-targeted photodynamic therapy for breast cancer

    doi: 10.1007/s10549-010-0957-1

    Figure Lengend Snippet: fVII is internalized by TF-expressing VEGF-stimulated angiogenic HUVECs and MDA-MB-231 breast cancer cells. fVII protein primarily bound to the cell plasma membrane ( green, arrows in a and d ) of the fixed cells (0 min), while endocytosis of fVII, shown as co-localization intracellularly with Alexa Fluor 633-phalloidin-stained cytoskeletal F-actin ( red, arrows in b and e ), was observed at 37°C for 30 min after incubation with VEGF-stimulated HUVECs ( b ) and human breast cancer MDA-MB-231 cells ( e ). c and f Secondary antibody FITC controls for non-specific FITC background. Scale bars , 20 µm

    Article Snippet: TF expression was induced on HUVECs using 1 nM VEGF (BD Biosciences) as previously described [ ].

    Techniques: Expressing, Multiple Displacement Amplification, Staining, Incubation

    fVII-tPDT selectively kills breast cancer cells ( a, b ) and angiogenic HUVECs ( d ) but has no side effects in normal cells such as 293 cells ( c ) and unstimulated HUVECs ( d ). a–c TF expression and selective effect of fVII-tPDT on human breast cancer MDA-MB-231 ( a ) and MCF-7 ( b ) cells and normal 293 cells ( c ). The level of TF expression was determined by flow cytometry using goat anti-HTF antibody ( solid line ). The control ( dashed line ) was incubated only with the secondary antibody FITC. The fVII-tPDT (2 µM SnCe6) treatment of these cell lines was performed with various laser fluence values, and efficacy was determined by crystal violet staining. d fVII-tPDT and ntPDT (2 µM SnCe6) treatment of angiogenic (VEGF-stimulated) and quiescent (unstimulated) HUVECs. a–d Representative of two experiments

    Journal: Breast cancer research and treatment

    Article Title: Selective and effective killing of angiogenic vascular endothelial cells and cancer cells by targeting tissue factor using a factor VII-targeted photodynamic therapy for breast cancer

    doi: 10.1007/s10549-010-0957-1

    Figure Lengend Snippet: fVII-tPDT selectively kills breast cancer cells ( a, b ) and angiogenic HUVECs ( d ) but has no side effects in normal cells such as 293 cells ( c ) and unstimulated HUVECs ( d ). a–c TF expression and selective effect of fVII-tPDT on human breast cancer MDA-MB-231 ( a ) and MCF-7 ( b ) cells and normal 293 cells ( c ). The level of TF expression was determined by flow cytometry using goat anti-HTF antibody ( solid line ). The control ( dashed line ) was incubated only with the secondary antibody FITC. The fVII-tPDT (2 µM SnCe6) treatment of these cell lines was performed with various laser fluence values, and efficacy was determined by crystal violet staining. d fVII-tPDT and ntPDT (2 µM SnCe6) treatment of angiogenic (VEGF-stimulated) and quiescent (unstimulated) HUVECs. a–d Representative of two experiments

    Article Snippet: TF expression was induced on HUVECs using 1 nM VEGF (BD Biosciences) as previously described [ ].

    Techniques: Expressing, Multiple Displacement Amplification, Flow Cytometry, Cytometry, Incubation, Staining

    Fluorescence images of VEGF IR in detrusor smooth muscle in the bladder neck region in whole-mount preparations of urinary bladder in control rats ( A and B ) and rats treated with CYP for 4 h ( C and D ) and 48 h ( E and F ). VEGF IR in the detrusor smooth

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    doi: 10.1152/ajprenal.90305.2008

    Figure Lengend Snippet: Fluorescence images of VEGF IR in detrusor smooth muscle in the bladder neck region in whole-mount preparations of urinary bladder in control rats ( A and B ) and rats treated with CYP for 4 h ( C and D ) and 48 h ( E and F ). VEGF IR in the detrusor smooth

    Article Snippet: Briefly, sections were incubated with 400 μl of rabbit anti-VEGF-A (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 1% goat serum and 0.1 M phosphate buffer overnight at room temperature.

    Techniques: Fluorescence

    VEGF immunoreactivity (IR) in cryostat sections of urothelium after CYP treatment. Urothelium (U) was outlined (green) and measured in total-pixels area ( A , D , G , and J ). A threshold encompassing an intensity range of 100–250 gray-scale values

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    doi: 10.1152/ajprenal.90305.2008

    Figure Lengend Snippet: VEGF immunoreactivity (IR) in cryostat sections of urothelium after CYP treatment. Urothelium (U) was outlined (green) and measured in total-pixels area ( A , D , G , and J ). A threshold encompassing an intensity range of 100–250 gray-scale values

    Article Snippet: Briefly, sections were incubated with 400 μl of rabbit anti-VEGF-A (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 1% goat serum and 0.1 M phosphate buffer overnight at room temperature.

    Techniques:

    Regulation of urinary bladder VEGF co-receptor neuropilin-1 (Npn-1) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    doi: 10.1152/ajprenal.90305.2008

    Figure Lengend Snippet: Regulation of urinary bladder VEGF co-receptor neuropilin-1 (Npn-1) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder

    Article Snippet: Briefly, sections were incubated with 400 μl of rabbit anti-VEGF-A (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 1% goat serum and 0.1 M phosphate buffer overnight at room temperature.

    Techniques: Reverse Transcription Polymerase Chain Reaction

    Regulation of urinary bladder VEGF co-receptor neuropilin-2 (Npn-2) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    doi: 10.1152/ajprenal.90305.2008

    Figure Lengend Snippet: Regulation of urinary bladder VEGF co-receptor neuropilin-2 (Npn-2) transcript level in control rats, after 2–6 h of CYP treatment, and after chronic (10 day) CYP-induced bladder inflammation. Top : semiquantitative RT-PCR showing urinary bladder

    Article Snippet: Briefly, sections were incubated with 400 μl of rabbit anti-VEGF-A (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 1% goat serum and 0.1 M phosphate buffer overnight at room temperature.

    Techniques: Reverse Transcription Polymerase Chain Reaction

    VEGF protein expression in the whole bladder with cyclophosphamide (CYP)-induced cystitis measured by ELISAs. Increases in VEGF protein expression were observed at all time points [2–48 h and 10 days (chronic)], but acute (2–4

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    doi: 10.1152/ajprenal.90305.2008

    Figure Lengend Snippet: VEGF protein expression in the whole bladder with cyclophosphamide (CYP)-induced cystitis measured by ELISAs. Increases in VEGF protein expression were observed at all time points [2–48 h and 10 days (chronic)], but acute (2–4

    Article Snippet: Briefly, sections were incubated with 400 μl of rabbit anti-VEGF-A (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 1% goat serum and 0.1 M phosphate buffer overnight at room temperature.

    Techniques: Expressing

    Semiquantitative analysis of VEGF IR in urothelium with CYP-induced cystitis. Values are means ± SE ( n = 6 for each group). * P ≤ 0.05.

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    doi: 10.1152/ajprenal.90305.2008

    Figure Lengend Snippet: Semiquantitative analysis of VEGF IR in urothelium with CYP-induced cystitis. Values are means ± SE ( n = 6 for each group). * P ≤ 0.05.

    Article Snippet: Briefly, sections were incubated with 400 μl of rabbit anti-VEGF-A (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 1% goat serum and 0.1 M phosphate buffer overnight at room temperature.

    Techniques:

    Fluorescence images of VEGF IR in suburothelial vasculature in the bladder neck region in whole-mount preparations of urinary bladder in control rats ( A and B ) and rats treated with CYP for 4 h ( C and D ) and 48 h ( E and F ). VEGF IR in the vasculature

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Upregulation of vascular endothelial growth factor isoform VEGF-164 and receptors (VEGFR-2, Npn-1, and Npn-2) in rats with cyclophosphamide-induced cystitis

    doi: 10.1152/ajprenal.90305.2008

    Figure Lengend Snippet: Fluorescence images of VEGF IR in suburothelial vasculature in the bladder neck region in whole-mount preparations of urinary bladder in control rats ( A and B ) and rats treated with CYP for 4 h ( C and D ) and 48 h ( E and F ). VEGF IR in the vasculature

    Article Snippet: Briefly, sections were incubated with 400 μl of rabbit anti-VEGF-A (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) in 1% goat serum and 0.1 M phosphate buffer overnight at room temperature.

    Techniques: Fluorescence

    Effects of Lenti-miRNA-VEGF-A and/or Lenti-miRNA-VEGF-C on cancer growth in GC bearing nude mice. A. Cancer volumes were significantly reduced in mice treated with Lenti-miRNA-VEGF-A, Lenti-miRNA-VEGF-C, or Lenti-miRNA-VEGF-A +VEGF-C, as compared to those

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Overexpression of both VEGF-A and VEGF-C in gastric cancer correlates with prognosis, and silencing of both is effective to inhibit cancer growth

    doi:

    Figure Lengend Snippet: Effects of Lenti-miRNA-VEGF-A and/or Lenti-miRNA-VEGF-C on cancer growth in GC bearing nude mice. A. Cancer volumes were significantly reduced in mice treated with Lenti-miRNA-VEGF-A, Lenti-miRNA-VEGF-C, or Lenti-miRNA-VEGF-A +VEGF-C, as compared to those

    Article Snippet: Western blotting was done using antibodies against VEGF-A and VEGF-C (Santa Cruz Biotechnology, CA, USA).

    Techniques: Mouse Assay

    Detection of expressions of VEGF-A and VEGF-C in GC by immunohistochemistry and double immunohistochemical staining for D2-40 and CD34. A. VEGF-A expression in the cytoplasm (×200). B. VEGF-C expression in the cytoplasm (×400). C. The

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Overexpression of both VEGF-A and VEGF-C in gastric cancer correlates with prognosis, and silencing of both is effective to inhibit cancer growth

    doi:

    Figure Lengend Snippet: Detection of expressions of VEGF-A and VEGF-C in GC by immunohistochemistry and double immunohistochemical staining for D2-40 and CD34. A. VEGF-A expression in the cytoplasm (×200). B. VEGF-C expression in the cytoplasm (×400). C. The

    Article Snippet: Western blotting was done using antibodies against VEGF-A and VEGF-C (Santa Cruz Biotechnology, CA, USA).

    Techniques: Immunohistochemistry, Staining, Expressing

    Combined silencing of VEGF-A and VEGF-C significantly inhibited cancer growth in vivo. A. SGC7901; B. Lenti-miRNA-neg; C. Lenti-miRNA-VEGF-A; D. Lenti-miRNA- VEGF-C; E. Lenti-miRNA-VEGF-A + Lenti-miRNA-VEGF-C. F. Silencing of VEGF-A or VEGF-C in SGC7901

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Overexpression of both VEGF-A and VEGF-C in gastric cancer correlates with prognosis, and silencing of both is effective to inhibit cancer growth

    doi:

    Figure Lengend Snippet: Combined silencing of VEGF-A and VEGF-C significantly inhibited cancer growth in vivo. A. SGC7901; B. Lenti-miRNA-neg; C. Lenti-miRNA-VEGF-A; D. Lenti-miRNA- VEGF-C; E. Lenti-miRNA-VEGF-A + Lenti-miRNA-VEGF-C. F. Silencing of VEGF-A or VEGF-C in SGC7901

    Article Snippet: Western blotting was done using antibodies against VEGF-A and VEGF-C (Santa Cruz Biotechnology, CA, USA).

    Techniques: In Vivo

    Combined silencing of both VEGF-A and VEGF-C significantly suppressed the cell proliferation and induced the apoptosis of GA cells. A. Cell proliferation was significantly inhibited in SGC7901 cells after infection with Lenti-miRNA-VEGF-A + Lenti-miRNA-VEGF-C

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Overexpression of both VEGF-A and VEGF-C in gastric cancer correlates with prognosis, and silencing of both is effective to inhibit cancer growth

    doi:

    Figure Lengend Snippet: Combined silencing of both VEGF-A and VEGF-C significantly suppressed the cell proliferation and induced the apoptosis of GA cells. A. Cell proliferation was significantly inhibited in SGC7901 cells after infection with Lenti-miRNA-VEGF-A + Lenti-miRNA-VEGF-C

    Article Snippet: Western blotting was done using antibodies against VEGF-A and VEGF-C (Santa Cruz Biotechnology, CA, USA).

    Techniques: Infection

    Expression of VEGF-A or VEGF-C in SGC7901 GC cells transduced with pCMV-VEGF-A miRNA or pCMV-VEGF-C miRNA expression plasmids at 48 h (*P

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Overexpression of both VEGF-A and VEGF-C in gastric cancer correlates with prognosis, and silencing of both is effective to inhibit cancer growth

    doi:

    Figure Lengend Snippet: Expression of VEGF-A or VEGF-C in SGC7901 GC cells transduced with pCMV-VEGF-A miRNA or pCMV-VEGF-C miRNA expression plasmids at 48 h (*P

    Article Snippet: Western blotting was done using antibodies against VEGF-A and VEGF-C (Santa Cruz Biotechnology, CA, USA).

    Techniques: Expressing, Transduction

    Relationship between expressions of VEGF-A and VEGF-C and overall survival.

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Overexpression of both VEGF-A and VEGF-C in gastric cancer correlates with prognosis, and silencing of both is effective to inhibit cancer growth

    doi:

    Figure Lengend Snippet: Relationship between expressions of VEGF-A and VEGF-C and overall survival.

    Article Snippet: Western blotting was done using antibodies against VEGF-A and VEGF-C (Santa Cruz Biotechnology, CA, USA).

    Techniques:

    Expression of Flag tag in hypoxic myocardium. The expression of Flag tag, which was fused with C-terminus of VEGF, was detected in ischemic myocardium by immunofluorescence staining. ( A ) Representative images for each condition is shown (scale bar = 50 μm). ( B ) The quantification of FITC fluorescence intensity was performed as described in Methods section. Results are presented as mean ± SEM (n = 8), ** p

    Journal: Scientific Reports

    Article Title: Gene delivery of hypoxia-inducible VEGF targeting collagen effectively improves cardiac function after myocardial infarction

    doi: 10.1038/s41598-017-13547-1

    Figure Lengend Snippet: Expression of Flag tag in hypoxic myocardium. The expression of Flag tag, which was fused with C-terminus of VEGF, was detected in ischemic myocardium by immunofluorescence staining. ( A ) Representative images for each condition is shown (scale bar = 50 μm). ( B ) The quantification of FITC fluorescence intensity was performed as described in Methods section. Results are presented as mean ± SEM (n = 8), ** p

    Article Snippet: Moreover, immunohistological staining was performed on the paraffin sections with anti-VEGF-A antibody (5 μg/mL, Abcam, USA) to evaluate the retained VEGF in infarct zone.

    Techniques: Expressing, FLAG-tag, Immunofluorescence, Staining, Fluorescence

    Expression of hVEGF in hypoxic myocardium and serum. ( A ) The expression of hVEGF in hypoxic myocardium was examined by immunohistochemistry using anti-human VEGF-A antibody. Representative images show increasing expression of hVEGF in mice treated with lentivirus expressing hVEGF, especially CBDhVEGF, compared with mice treated with control virus (scale bar = 50 μm). ( B ) Human VEGF production in serum was examined by a human VEGF ELISA assay. Results are presented as mean ± SEM (n = 8), *** p

    Journal: Scientific Reports

    Article Title: Gene delivery of hypoxia-inducible VEGF targeting collagen effectively improves cardiac function after myocardial infarction

    doi: 10.1038/s41598-017-13547-1

    Figure Lengend Snippet: Expression of hVEGF in hypoxic myocardium and serum. ( A ) The expression of hVEGF in hypoxic myocardium was examined by immunohistochemistry using anti-human VEGF-A antibody. Representative images show increasing expression of hVEGF in mice treated with lentivirus expressing hVEGF, especially CBDhVEGF, compared with mice treated with control virus (scale bar = 50 μm). ( B ) Human VEGF production in serum was examined by a human VEGF ELISA assay. Results are presented as mean ± SEM (n = 8), *** p

    Article Snippet: Moreover, immunohistological staining was performed on the paraffin sections with anti-VEGF-A antibody (5 μg/mL, Abcam, USA) to evaluate the retained VEGF in infarct zone.

    Techniques: Expressing, Immunohistochemistry, Mouse Assay, Enzyme-linked Immunosorbent Assay