vegf a  (Millipore)


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    Name:
    VEGF human
    Description:
    VEGF vascular endothelial growth factor signals through the three receptors fms like tyrosine kinase flt 1 KDR gene product the murine homolog of KDR is the flk 1 gene product and the flt4 gene product Recombinant human VEGF is a 38 2kDa disulfide linked homodimeric protein consisting of two 165 amino acid polypeptide chains
    Catalog Number:
    srp3182
    Price:
    None
    Applications:
    VEGF human has been used as a chemoattractant for HUVECs (human umbilical vein endothelial cells) migration assay.
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    Structured Review

    Millipore vegf a
    VEGF human
    VEGF vascular endothelial growth factor signals through the three receptors fms like tyrosine kinase flt 1 KDR gene product the murine homolog of KDR is the flk 1 gene product and the flt4 gene product Recombinant human VEGF is a 38 2kDa disulfide linked homodimeric protein consisting of two 165 amino acid polypeptide chains
    https://www.bioz.com/result/vegf a/product/Millipore
    Average 99 stars, based on 50 article reviews
    Price from $9.99 to $1999.99
    vegf a - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells"

    Article Title: Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0148968

    Effects of ABL on VEGF-induced HUVEC migration and tube formation. A-B) ABL increases VEGF-induced ECs transwell migration. The data represent the number of migrated cells per microscopic field in VEGF-treated cells vs. control cells. C-D) ABL increases VEGF-induced EC transwell migration. Confluent HUVECs in a monolayer were pretreated with ABL or vehicle for 2 h and wounded with a cell scraper. After 48 h of incubation at 37°C, the number of cells that migrated across the wound edge was counted in each field. E-F) ABL increases VEGF-induced EC tube formation. The images were visualized using a phase contrast microscope (10× magnification). The total tubule length from 10 non-overlapping fields was measured by tracing the tube-like structure. The values represent the mean ± SEM from 3 independent experiments ( n = 3). Scale bar: 100 μm.
    Figure Legend Snippet: Effects of ABL on VEGF-induced HUVEC migration and tube formation. A-B) ABL increases VEGF-induced ECs transwell migration. The data represent the number of migrated cells per microscopic field in VEGF-treated cells vs. control cells. C-D) ABL increases VEGF-induced EC transwell migration. Confluent HUVECs in a monolayer were pretreated with ABL or vehicle for 2 h and wounded with a cell scraper. After 48 h of incubation at 37°C, the number of cells that migrated across the wound edge was counted in each field. E-F) ABL increases VEGF-induced EC tube formation. The images were visualized using a phase contrast microscope (10× magnification). The total tubule length from 10 non-overlapping fields was measured by tracing the tube-like structure. The values represent the mean ± SEM from 3 independent experiments ( n = 3). Scale bar: 100 μm.

    Techniques Used: Migration, Incubation, Microscopy

    Schematic representation of the ABL effect on VEGF signaling in ECs. ABL inhibits VEGFR-2 and VE-cadherin association, thus promoting VEGFR-2 internalization, following by VEGF-induced VEGFR-2 phosphorylation and downstream signaling.
    Figure Legend Snippet: Schematic representation of the ABL effect on VEGF signaling in ECs. ABL inhibits VEGFR-2 and VE-cadherin association, thus promoting VEGFR-2 internalization, following by VEGF-induced VEGFR-2 phosphorylation and downstream signaling.

    Techniques Used:

    ABL enhances VEGF signaling in endothelial cells. A) HUVECs were serum-starved overnight and pretreated with ABL or vehicle for 2 h, followed by exposure to VEGF-A (50 ng/mL) for various times as indicated. MAPK MAPK p44/42 Thr202/Tyr204 , p38 Thr180/Tyr182 , and Akt Ser473 phosphorylation in the cell lysates were measured via western blot analysis. B-D) The quantitative analysis represents the ratio of phosphorylated/total MAPK MAPK p44/42, MAPK p38, and Akt from 3 independent experiments. E) VEGFR-2 Tyr1175 phosphorylation in the cell lysates was measured via western blot analysis. F) Quantitative analysis of the ratio of phosphorylated/total VEGFR-2 from three independent experiments ( n = 3).
    Figure Legend Snippet: ABL enhances VEGF signaling in endothelial cells. A) HUVECs were serum-starved overnight and pretreated with ABL or vehicle for 2 h, followed by exposure to VEGF-A (50 ng/mL) for various times as indicated. MAPK MAPK p44/42 Thr202/Tyr204 , p38 Thr180/Tyr182 , and Akt Ser473 phosphorylation in the cell lysates were measured via western blot analysis. B-D) The quantitative analysis represents the ratio of phosphorylated/total MAPK MAPK p44/42, MAPK p38, and Akt from 3 independent experiments. E) VEGFR-2 Tyr1175 phosphorylation in the cell lysates was measured via western blot analysis. F) Quantitative analysis of the ratio of phosphorylated/total VEGFR-2 from three independent experiments ( n = 3).

    Techniques Used: Western Blot

    ABL increases VEGF-induced HUVEC growth and proliferation. A) VEGF-induced EC growth monitored by the MTT assay following treatment with different doses of ABL or vehicle. The data represent the percent increase after 48 h relative to non-stimulated cells from 3 independent experiments. B) VEGF-induced [ 3 H]-thymidine incorporation following different doses of ABL or vehicle. The data represent the percent increase after 48 h relative to the non-stimulated cells from 3 independent experiments ( n = 3). * P
    Figure Legend Snippet: ABL increases VEGF-induced HUVEC growth and proliferation. A) VEGF-induced EC growth monitored by the MTT assay following treatment with different doses of ABL or vehicle. The data represent the percent increase after 48 h relative to non-stimulated cells from 3 independent experiments. B) VEGF-induced [ 3 H]-thymidine incorporation following different doses of ABL or vehicle. The data represent the percent increase after 48 h relative to the non-stimulated cells from 3 independent experiments ( n = 3). * P

    Techniques Used: MTT Assay

    ABL enhances VEGFR-2 internalization in ECs. A) Colocalization of VEGFR-2 with endosome markers EEA1 in ECs. The ECs were pretreated with ABL for 2 h following fifteen minutes of VEGF stimulation, fixed, permeablized, and labeled with anti-VEGFR-2 (red) and anti-EEA1 (green) and processed for confocal microscopy. The endocytic trafficking of VEGFR2 without locating in endosome was observed in ECs (arrow). B) Colocalization of VEGFR-2 (red) and VE-cadherin (green) was observed in ECs (arrow). Nuclei were counterstained with DAPI (blue). Scale bar = 20 μm.
    Figure Legend Snippet: ABL enhances VEGFR-2 internalization in ECs. A) Colocalization of VEGFR-2 with endosome markers EEA1 in ECs. The ECs were pretreated with ABL for 2 h following fifteen minutes of VEGF stimulation, fixed, permeablized, and labeled with anti-VEGFR-2 (red) and anti-EEA1 (green) and processed for confocal microscopy. The endocytic trafficking of VEGFR2 without locating in endosome was observed in ECs (arrow). B) Colocalization of VEGFR-2 (red) and VE-cadherin (green) was observed in ECs (arrow). Nuclei were counterstained with DAPI (blue). Scale bar = 20 μm.

    Techniques Used: Labeling, Confocal Microscopy

    ABL enhances Matrigel angiogenesis in vivo . A) Examples of CD31 immunofluorescence staining of Matrigel plugs containing VEGF-A or control buffer. B) The quantification data represent the percent increase in CD31-positive area in comparison with the control sample ( n = 6). C) Quantitative analysis of GAPDH-normalized VE-cadherin ( Cdh5 ) mRNA expression in Matrigel plugs containing VEGF-A or control buffer that were implanted in mice ( n = 3). Scale bars: 100 μm.
    Figure Legend Snippet: ABL enhances Matrigel angiogenesis in vivo . A) Examples of CD31 immunofluorescence staining of Matrigel plugs containing VEGF-A or control buffer. B) The quantification data represent the percent increase in CD31-positive area in comparison with the control sample ( n = 6). C) Quantitative analysis of GAPDH-normalized VE-cadherin ( Cdh5 ) mRNA expression in Matrigel plugs containing VEGF-A or control buffer that were implanted in mice ( n = 3). Scale bars: 100 μm.

    Techniques Used: In Vivo, Immunofluorescence, Staining, Expressing, Mouse Assay

    2) Product Images from "Hypericin-photodynamic therapy induces human umbilical vein endothelial cell apoptosis"

    Article Title: Hypericin-photodynamic therapy induces human umbilical vein endothelial cell apoptosis

    Journal: Scientific Reports

    doi: 10.1038/srep18398

    Inhibition of VEGF-A-mediated PI3K/Akt pathway in HUVECs. The HUVECs were serum-starved for 24 h and stimulated in fresh medium with VEGF-A (25 ng/ml) and then treated with HY (0.062 μM) in combination with VEGF-A (25 ng/ml) for 24 h. The cells were exposed to a 585-nm LED light at a dose of 1.0 J/cm 2 and were incubation for 24 h. The cells were lysed and the proteins were harvested for western blot analysis of VEGF-A ( a ), p-Akt (Ser473) and Akt ( b ), and Bad ( c ). Densitometric measurements were analysed using AlphaEaseFC 4.0 software. The protein expression levels were normalized to those of the vehicle control (100%). Data are presented as means ± S.D. (n = 3); ** P
    Figure Legend Snippet: Inhibition of VEGF-A-mediated PI3K/Akt pathway in HUVECs. The HUVECs were serum-starved for 24 h and stimulated in fresh medium with VEGF-A (25 ng/ml) and then treated with HY (0.062 μM) in combination with VEGF-A (25 ng/ml) for 24 h. The cells were exposed to a 585-nm LED light at a dose of 1.0 J/cm 2 and were incubation for 24 h. The cells were lysed and the proteins were harvested for western blot analysis of VEGF-A ( a ), p-Akt (Ser473) and Akt ( b ), and Bad ( c ). Densitometric measurements were analysed using AlphaEaseFC 4.0 software. The protein expression levels were normalized to those of the vehicle control (100%). Data are presented as means ± S.D. (n = 3); ** P

    Techniques Used: Inhibition, Incubation, Western Blot, Software, Expressing

    3) Product Images from "A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC-? and DNA synthesis in vascular endothelial cells"

    Article Title: A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC-? and DNA synthesis in vascular endothelial cells

    Journal: The EMBO Journal

    doi: 10.1093/emboj/20.11.2768

    Fig. 8. The C-terminal SH2 domain of PLC-γ associates with KDR/Flk-1 through phosphorylated Y1175. ( A ) An in vivo association of KDR/Flk-1 with PLC-γ. BAE cells were infected with adenovirus vectors containing wild-type or Y1175F. After stimulation with VEGF-A (10 ng/ml), cell lysates were prepared and immunoprecipitated with anti-KDR/Flk-1 antibody. The samples were resolved by SDS–PAGE and immunoblotted with anti-PLC-γ antibodies or anti-KDR/Flk-1 as indicated. (B–E) In vitro association of KDR/Flk-1 with PLC-γ. NIH-3T3-KDR cell lysates unstimulated or stimulated with VEGF-A (10 ng/ml) were incubated with 1.5 µg of GST, GST–PLC-γ SH2–SH2, GST–PLC-γ N-SH2 or GST–PLC-γ C-SH2 protein pre-bound to glutathione–Sepharose beads for 2 h at 4°C ( B ). The same experiments were carried out using GST, GST–PLC-γ C-SH2, GST–Grb2 SH2 or GST–PI3 kinase N-SH2 protein ( C ). The same cell lysates as in (A) were incubated with 1.5 µg of GST–PLC-γ C-SH2 protein pre-bound to glutathione–Sepharose beads. During the incubation, antibody ( D ) or peptide ( E ) was added as a competitor. The beads were then washed three times with cold lysis buffer. The proteins bound to the beads or total cell lysate (TCL) were resolved by SDS–PAGE and immunoblotted with anti-KDR/Flk-1 or anti-GST antibody as shown.
    Figure Legend Snippet: Fig. 8. The C-terminal SH2 domain of PLC-γ associates with KDR/Flk-1 through phosphorylated Y1175. ( A ) An in vivo association of KDR/Flk-1 with PLC-γ. BAE cells were infected with adenovirus vectors containing wild-type or Y1175F. After stimulation with VEGF-A (10 ng/ml), cell lysates were prepared and immunoprecipitated with anti-KDR/Flk-1 antibody. The samples were resolved by SDS–PAGE and immunoblotted with anti-PLC-γ antibodies or anti-KDR/Flk-1 as indicated. (B–E) In vitro association of KDR/Flk-1 with PLC-γ. NIH-3T3-KDR cell lysates unstimulated or stimulated with VEGF-A (10 ng/ml) were incubated with 1.5 µg of GST, GST–PLC-γ SH2–SH2, GST–PLC-γ N-SH2 or GST–PLC-γ C-SH2 protein pre-bound to glutathione–Sepharose beads for 2 h at 4°C ( B ). The same experiments were carried out using GST, GST–PLC-γ C-SH2, GST–Grb2 SH2 or GST–PI3 kinase N-SH2 protein ( C ). The same cell lysates as in (A) were incubated with 1.5 µg of GST–PLC-γ C-SH2 protein pre-bound to glutathione–Sepharose beads. During the incubation, antibody ( D ) or peptide ( E ) was added as a competitor. The beads were then washed three times with cold lysis buffer. The proteins bound to the beads or total cell lysate (TCL) were resolved by SDS–PAGE and immunoblotted with anti-KDR/Flk-1 or anti-GST antibody as shown.

    Techniques Used: Planar Chromatography, In Vivo, Infection, Immunoprecipitation, SDS Page, In Vitro, Incubation, Lysis

    Fig. 6. Mutation of Y1175F in KDR/Flk-1 blocks VEGF-A-induced initiation of DNA synthesis. BAE cells were infected with adenovirus vectors containing wild-type or mutant receptors and subsequently starved for 48 h in DMEM–0.1% FCS. The total cell lysates were blotted with anti-KDR/Flk-1 antibody. NIH-3T3-KDR cells were used as the positive control ( A ). Alternatively, BAE cells were stimulated with BSA or VEGF-A (10 ng/ml). After incubation for 16 h, cells were pulse-labeled with [ 3 H]thymidine (1 mCi/ml) for 4 h. Subsequently, they were harvested on glass filters and incorporated radioactivity was measured. Data represent the average of triplicate samples. Fold induction was calculated relative to the values for the cells stimulated by BSA in each mutant ( B ).
    Figure Legend Snippet: Fig. 6. Mutation of Y1175F in KDR/Flk-1 blocks VEGF-A-induced initiation of DNA synthesis. BAE cells were infected with adenovirus vectors containing wild-type or mutant receptors and subsequently starved for 48 h in DMEM–0.1% FCS. The total cell lysates were blotted with anti-KDR/Flk-1 antibody. NIH-3T3-KDR cells were used as the positive control ( A ). Alternatively, BAE cells were stimulated with BSA or VEGF-A (10 ng/ml). After incubation for 16 h, cells were pulse-labeled with [ 3 H]thymidine (1 mCi/ml) for 4 h. Subsequently, they were harvested on glass filters and incorporated radioactivity was measured. Data represent the average of triplicate samples. Fold induction was calculated relative to the values for the cells stimulated by BSA in each mutant ( B ).

    Techniques Used: Mutagenesis, DNA Synthesis, Infection, Positive Control, Incubation, Labeling, Radioactivity

    Fig. 9. Inhibition of VEGF-A-induced DNA synthesis in SE cells. SE cells were starved in modified HuMedia-EG2 medium without VEGF-A for 6–7 h. The cells were either not injected ( A ), injected with control normal rabbit IgG or injected with anti-PY1175 antibody ( B ). After 1–3 h, the injected cells were stimulated with VEGF-A and simultaneously labeled with BrdU for 20 h. The injected cells were identified by staining with FITC-labeled anti-rabbit antibody and BrdU-incorporated cells by staining with anti-BrdU antibody. Results were expressed as the percentage of stained cells exhibiting nuclear fluorescence ( C ).
    Figure Legend Snippet: Fig. 9. Inhibition of VEGF-A-induced DNA synthesis in SE cells. SE cells were starved in modified HuMedia-EG2 medium without VEGF-A for 6–7 h. The cells were either not injected ( A ), injected with control normal rabbit IgG or injected with anti-PY1175 antibody ( B ). After 1–3 h, the injected cells were stimulated with VEGF-A and simultaneously labeled with BrdU for 20 h. The injected cells were identified by staining with FITC-labeled anti-rabbit antibody and BrdU-incorporated cells by staining with anti-BrdU antibody. Results were expressed as the percentage of stained cells exhibiting nuclear fluorescence ( C ).

    Techniques Used: Inhibition, DNA Synthesis, Modification, Injection, Labeling, Staining, Fluorescence

    Fig. 2. The in vitro kinase assay, phosphoamino acid analysis and phosphopeptide mapping of KDR/Flk-1 overexpressed in NIH-3T3 and HUVE cells. ( A ) The in vitro kinase assay in KDR/Flk-1. The wild-type KDR/Flk-1 was immunoprecipitated from unstimulated or VEGF-A-stimulated NIH-3T3-KDR or HUVEC-KDR cells, and labeled in the in vitro kinase assay in the presence of [γ- 32 P]ATP. The radiolabeled KDR/Flk-1 was subjected to SDS–PAGE and autoradio graphy. ( B ) Phosphoamino acid analysis. The radiolabeled KDR/Flk-1 band was excised from the gel and subjected to trypsin digestion. The resulting phosphopeptides were then hydrolyzed and analyzed by two-dimensional chromatography (see Materials and methods). ( C ) Tryptic phosphopeptide mapping of KDR/Flk-1. The KDR/Flk-1 was labeled by the in vitro kinase assay and subjected to SDS–PAGE. The labeled KDR/Flk-1 band as shown in (A) was excised from the gel and subjected to trypsin digestion. The resultant phosphopeptides were resolved by electrophoresis at pH 8.9, followed by thin-layer chromatography (see Materials and methods).
    Figure Legend Snippet: Fig. 2. The in vitro kinase assay, phosphoamino acid analysis and phosphopeptide mapping of KDR/Flk-1 overexpressed in NIH-3T3 and HUVE cells. ( A ) The in vitro kinase assay in KDR/Flk-1. The wild-type KDR/Flk-1 was immunoprecipitated from unstimulated or VEGF-A-stimulated NIH-3T3-KDR or HUVEC-KDR cells, and labeled in the in vitro kinase assay in the presence of [γ- 32 P]ATP. The radiolabeled KDR/Flk-1 was subjected to SDS–PAGE and autoradio graphy. ( B ) Phosphoamino acid analysis. The radiolabeled KDR/Flk-1 band was excised from the gel and subjected to trypsin digestion. The resulting phosphopeptides were then hydrolyzed and analyzed by two-dimensional chromatography (see Materials and methods). ( C ) Tryptic phosphopeptide mapping of KDR/Flk-1. The KDR/Flk-1 was labeled by the in vitro kinase assay and subjected to SDS–PAGE. The labeled KDR/Flk-1 band as shown in (A) was excised from the gel and subjected to trypsin digestion. The resultant phosphopeptides were resolved by electrophoresis at pH 8.9, followed by thin-layer chromatography (see Materials and methods).

    Techniques Used: In Vitro, Kinase Assay, Phosphoamino Acid Analysis, Immunoprecipitation, Labeling, SDS Page, Two-dimensional Chromatography, Electrophoresis, Thin Layer Chromatography

    Fig. 7. In vivo Y1175 phosphorylation in VEGF-A-stimulated cells. MSS cells were infected with adenovirus vectors containing wild-type, Y1175F, Y1214F or Y801F. After 2 days, cells were stimulated with or without VEGF-A ( A ). NIH-3T3-KDR, NIH-3T3-Flt or HeLa cells were stimulated with VEGF-A, bFGF, PDGF or EGF as indicated ( B ). Primary endothelial cells, HUVE cells or rat SE cells were stimulated with VEGF-A, bFGF or HGF ( C ). Total cell lysates were blotted with anti-PY1175 (upper panel), anti-phosphotyrosine (middle panel) or anti-KDR/Flk-1 (lower panel) antibody. Histocytochemistry of HUVE cells using anti-PY1175. HUVE cells were starved and stimulated with VEGF-A, bFGF or PDGF for the indicated times. Subsequently, the cells were fixed and immunostained with anti-PY1175. Alternatively, PY1175 peptide was added as a competitor. The cells were examined with a fluorescent microscope ( D ). Bar, 50 µm.
    Figure Legend Snippet: Fig. 7. In vivo Y1175 phosphorylation in VEGF-A-stimulated cells. MSS cells were infected with adenovirus vectors containing wild-type, Y1175F, Y1214F or Y801F. After 2 days, cells were stimulated with or without VEGF-A ( A ). NIH-3T3-KDR, NIH-3T3-Flt or HeLa cells were stimulated with VEGF-A, bFGF, PDGF or EGF as indicated ( B ). Primary endothelial cells, HUVE cells or rat SE cells were stimulated with VEGF-A, bFGF or HGF ( C ). Total cell lysates were blotted with anti-PY1175 (upper panel), anti-phosphotyrosine (middle panel) or anti-KDR/Flk-1 (lower panel) antibody. Histocytochemistry of HUVE cells using anti-PY1175. HUVE cells were starved and stimulated with VEGF-A, bFGF or PDGF for the indicated times. Subsequently, the cells were fixed and immunostained with anti-PY1175. Alternatively, PY1175 peptide was added as a competitor. The cells were examined with a fluorescent microscope ( D ). Bar, 50 µm.

    Techniques Used: In Vivo, Infection, Microscopy

    Fig. 4. Analysis of KDR/Flk-1 mutants expressed in the adenovirus vector expression system. MSS31 cells were infected with adenovirus vectors containing wild-type or mutant KDR/Flk-1 receptors. Two days later, the cells were first starved for 12 h in DMEM–0.1% FCS and subsequently were not stimulated or stimulated with VEGF-A (10 ng/ml). Total cell lysates expressing the wild-type or various mutants of KDR/Flk-1 were analyzed by western blotting using anti-KDR/Flk-1 ( A ) or anti-phosphotyrosine ( B ) antibody. ( C ) The cell lysates were immunoprecipitated with anti-PLC-γ antibodies and blotted with anti-phosphotyrosine or anti-PLC-γ antibodies. ( D ) The same total cell lysates were blotted with anti-phosphoMAP kinase or anti-MAP kinase antibody. HUVE cells were used as the positive control.
    Figure Legend Snippet: Fig. 4. Analysis of KDR/Flk-1 mutants expressed in the adenovirus vector expression system. MSS31 cells were infected with adenovirus vectors containing wild-type or mutant KDR/Flk-1 receptors. Two days later, the cells were first starved for 12 h in DMEM–0.1% FCS and subsequently were not stimulated or stimulated with VEGF-A (10 ng/ml). Total cell lysates expressing the wild-type or various mutants of KDR/Flk-1 were analyzed by western blotting using anti-KDR/Flk-1 ( A ) or anti-phosphotyrosine ( B ) antibody. ( C ) The cell lysates were immunoprecipitated with anti-PLC-γ antibodies and blotted with anti-phosphotyrosine or anti-PLC-γ antibodies. ( D ) The same total cell lysates were blotted with anti-phosphoMAP kinase or anti-MAP kinase antibody. HUVE cells were used as the positive control.

    Techniques Used: Plasmid Preparation, Expressing, Infection, Mutagenesis, Western Blot, Immunoprecipitation, Planar Chromatography, Positive Control

    4) Product Images from "Lysosomal Pathways and Autophagy Distinctively Control Endothelial Cell Behavior to Affect Tumor Vasculature"

    Article Title: Lysosomal Pathways and Autophagy Distinctively Control Endothelial Cell Behavior to Affect Tumor Vasculature

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2019.00171

    CQ reduces proliferation and spheroid sprouting after VEGF-A stimulation. HUVEC proliferation was assessed based on confluency measurements in an IncuCyte imaging system. (A) From start of measurements at t = 0 (the moment of VEGF-A supplementation with indicated doses) images were taken every 2 h up to 48 h. Graphs display confluency results of the indicated conditions ( n = 4). At t = 48 h: (ii) p -value
    Figure Legend Snippet: CQ reduces proliferation and spheroid sprouting after VEGF-A stimulation. HUVEC proliferation was assessed based on confluency measurements in an IncuCyte imaging system. (A) From start of measurements at t = 0 (the moment of VEGF-A supplementation with indicated doses) images were taken every 2 h up to 48 h. Graphs display confluency results of the indicated conditions ( n = 4). At t = 48 h: (ii) p -value

    Techniques Used: Imaging

    CQ treatment, but not ATG5 deficiency, reduces VEGFR2 phosphorylation after VEGF-A stimulation. HUVECs were either (A–D) pretreated with CQ or (E-I) siRNA against ATG5 (siATG5) and treated with 50 ng/ml VEGF-A where indicated. Cells were treated with non-targeting siRNA (siScrambled; siScr) as control. (A,E,I) Cell lysates were analyzed by immunoblotting. (B,F) Graphs display (relative) band intensity ratio of phosphorylated or (C,G) p130 VEGFR2 to p250/p230 VEGFR2. (D,G) Bar graphs display VEGFR1 intensity normalized to GAPDH. (I) HUVECs were cultured in presence or absence of 25 μM CQ for 2 h. A short-term exposure was opted to minimalize any secondary effects on autophagy flux due to CQ treatment. N.D., not detected. (B–D,F–H) , n = 3, mean ± SEM.
    Figure Legend Snippet: CQ treatment, but not ATG5 deficiency, reduces VEGFR2 phosphorylation after VEGF-A stimulation. HUVECs were either (A–D) pretreated with CQ or (E-I) siRNA against ATG5 (siATG5) and treated with 50 ng/ml VEGF-A where indicated. Cells were treated with non-targeting siRNA (siScrambled; siScr) as control. (A,E,I) Cell lysates were analyzed by immunoblotting. (B,F) Graphs display (relative) band intensity ratio of phosphorylated or (C,G) p130 VEGFR2 to p250/p230 VEGFR2. (D,G) Bar graphs display VEGFR1 intensity normalized to GAPDH. (I) HUVECs were cultured in presence or absence of 25 μM CQ for 2 h. A short-term exposure was opted to minimalize any secondary effects on autophagy flux due to CQ treatment. N.D., not detected. (B–D,F–H) , n = 3, mean ± SEM.

    Techniques Used: Cell Culture

    5) Product Images from "Endogenous Angiogenesis Inhibitor Vasohibin1 Exhibits Broad-Spectrum Antilymphangiogenic Activity and Suppresses Lymph Node Metastasis"

    Article Title: Endogenous Angiogenesis Inhibitor Vasohibin1 Exhibits Broad-Spectrum Antilymphangiogenic Activity and Suppresses Lymph Node Metastasis

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2010.090829

    VASH1 inhibits angiogenesis and lymphangiogenesis induced by VEGF-A. A: Pellets containing 160 ng of VEGF-A plus or minus 4 ng of VASH1 were inoculated into the mouse cornea. Fourteen days after the inoculation, the corneas were harvested and immunostained
    Figure Legend Snippet: VASH1 inhibits angiogenesis and lymphangiogenesis induced by VEGF-A. A: Pellets containing 160 ng of VEGF-A plus or minus 4 ng of VASH1 were inoculated into the mouse cornea. Fourteen days after the inoculation, the corneas were harvested and immunostained

    Techniques Used:

    6) Product Images from "Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents"

    Article Title: Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents

    Journal: Nature Communications

    doi: 10.1038/ncomms15623

    Surface permeability of BBB spheroid to high molecular weight dextran and intact tight junctions are modulated by VEGF. ( a ) Fluorescence images showing the expression of tight junction markers, claudin 5 and occludin (white). Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar, 100 μm (× 20 objective). ( b ) Magnified fluorescence images showing claudin 5 and occluding expression. Scale bar, 100 μm (× 60 objective). ( c ) Fluorescence images showing decreased expression of tight junction marker (ZO-1: green) with increasing VEGF-A concentration (at 5, 20 and 50 ng ml −1 ) in primary HBMEC (pre-labelled with CellTracker Red dye) and ( d ) immortalized hCMEC/D3 ECs. Cell nuclei were labelled with Hoechst dye (blue). Scale bar: 50 μm in lower-magnification images; 10 μm in magnified images. ( e ) Dextran permeability assay showing that VEGF-A (at 25, 50 and 100 ng ml −1 ) increased spheroid permeability to TRITC-Dextran (155 kDa; red; 10 mg ml −1 ) using spheroids established using primary HBMEC ECs. The image panels above the graph depict a representation of how permeability was assessed. The white dotted line marks the area within the core of the spheroid, where the mean fluorescence intensity was quantified. Scale bar, 50 μm; n =8. ( f ) Dextran permeability study (as in e ) using spheroids established using immortalized hCMEC/D3 ECs. n spheroid =3–5. Both graphs show mean TRITC fluorescence intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars (** P
    Figure Legend Snippet: Surface permeability of BBB spheroid to high molecular weight dextran and intact tight junctions are modulated by VEGF. ( a ) Fluorescence images showing the expression of tight junction markers, claudin 5 and occludin (white). Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar, 100 μm (× 20 objective). ( b ) Magnified fluorescence images showing claudin 5 and occluding expression. Scale bar, 100 μm (× 60 objective). ( c ) Fluorescence images showing decreased expression of tight junction marker (ZO-1: green) with increasing VEGF-A concentration (at 5, 20 and 50 ng ml −1 ) in primary HBMEC (pre-labelled with CellTracker Red dye) and ( d ) immortalized hCMEC/D3 ECs. Cell nuclei were labelled with Hoechst dye (blue). Scale bar: 50 μm in lower-magnification images; 10 μm in magnified images. ( e ) Dextran permeability assay showing that VEGF-A (at 25, 50 and 100 ng ml −1 ) increased spheroid permeability to TRITC-Dextran (155 kDa; red; 10 mg ml −1 ) using spheroids established using primary HBMEC ECs. The image panels above the graph depict a representation of how permeability was assessed. The white dotted line marks the area within the core of the spheroid, where the mean fluorescence intensity was quantified. Scale bar, 50 μm; n =8. ( f ) Dextran permeability study (as in e ) using spheroids established using immortalized hCMEC/D3 ECs. n spheroid =3–5. Both graphs show mean TRITC fluorescence intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars (** P

    Techniques Used: Permeability, Molecular Weight, Fluorescence, Expressing, Staining, Marker, Concentration Assay, FITC-Dextran Permeability Assay

    7) Product Images from "High VEGF-D and Low MMP-2 Serum Levels Predict Nodal-Positive Disease in Invasive Bladder Cancer"

    Article Title: High VEGF-D and Low MMP-2 Serum Levels Predict Nodal-Positive Disease in Invasive Bladder Cancer

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

    doi: 10.12659/MSM.894383

    Markers were measured in serum of patients with invasive UCB (n=66) and in patients with pTa UCB or no history of UCB (n=22). For VEGF-A, only 85 samples could be evaluated. Dots represent patient samples. Horizontal lines represent mean values. *** p
    Figure Legend Snippet: Markers were measured in serum of patients with invasive UCB (n=66) and in patients with pTa UCB or no history of UCB (n=22). For VEGF-A, only 85 samples could be evaluated. Dots represent patient samples. Horizontal lines represent mean values. *** p

    Techniques Used:

    8) Product Images from "Increased Expression of Angiogenic Genes in the Brains of Mouse Meg3-Null Embryos"

    Article Title: Increased Expression of Angiogenic Genes in the Brains of Mouse Meg3-Null Embryos

    Journal: Endocrinology

    doi: 10.1210/en.2009-1151

    Simplified schematic representation of VEGF and Notch signaling cascades showing genes with expression change on the microarray and validated by quantitative RT-PCR. Genes whose increased expression predicted by the microarray analysis was validated by
    Figure Legend Snippet: Simplified schematic representation of VEGF and Notch signaling cascades showing genes with expression change on the microarray and validated by quantitative RT-PCR. Genes whose increased expression predicted by the microarray analysis was validated by

    Techniques Used: Expressing, Microarray, Quantitative RT-PCR

    9) Product Images from "Cell autonomous ANTXR1-mediated regulation of extracellular matrix components in primary fibroblasts"

    Article Title: Cell autonomous ANTXR1-mediated regulation of extracellular matrix components in primary fibroblasts

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    doi: 10.1016/j.matbio.2016.12.002

    VEGF induced transcript levels of collagen α1(I) and fibronectin. (A) VEGF protein levels were assessed by ELISA (*P
    Figure Legend Snippet: VEGF induced transcript levels of collagen α1(I) and fibronectin. (A) VEGF protein levels were assessed by ELISA (*P

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Mutant fibroblasts express high VEGF levels. (A) Bar graphs show average transcript levels (fold increase) in control and mutant fibroblasts isolated from P49 mice. (*P
    Figure Legend Snippet: Mutant fibroblasts express high VEGF levels. (A) Bar graphs show average transcript levels (fold increase) in control and mutant fibroblasts isolated from P49 mice. (*P

    Techniques Used: Mutagenesis, Isolation, Mouse Assay

    VEGF regulates collagen α1(I) and Fn1 protein levels. (A, B) Representative immunofluorescence images and data analyses of cells treated with 1μg/ml of VEGF neutralizing antibody or with 25 ng/ml of rVEGF. (n= 3; *, # , P
    Figure Legend Snippet: VEGF regulates collagen α1(I) and Fn1 protein levels. (A, B) Representative immunofluorescence images and data analyses of cells treated with 1μg/ml of VEGF neutralizing antibody or with 25 ng/ml of rVEGF. (n= 3; *, # , P

    Techniques Used: Immunofluorescence

    10) Product Images from "Anosmin-1 activates vascular endothelial growth factor receptor and its related signaling pathway for olfactory bulb angiogenesis"

    Article Title: Anosmin-1 activates vascular endothelial growth factor receptor and its related signaling pathway for olfactory bulb angiogenesis

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-57040-3

    Effects of anosmin-1 deletion mutants on VEGFR2 activation and cell migration. ( a ) Expression of VEGFR2 in PAE and PAE/KDR cells. Cell lysates were analyzed by western blotting with the anti-VEGFR2 and anti-β-actin Abs. ( b ) Transwell cell migration assay. PAE or PAE/KDR cells were seeded in the upper compartment, and were treated with the indicated concentrations of anosmin-1 or VEGF-A, or without anosmin-1 (Control). The cells that moved into the lower chamber were counted in five different microscopic fields. **P
    Figure Legend Snippet: Effects of anosmin-1 deletion mutants on VEGFR2 activation and cell migration. ( a ) Expression of VEGFR2 in PAE and PAE/KDR cells. Cell lysates were analyzed by western blotting with the anti-VEGFR2 and anti-β-actin Abs. ( b ) Transwell cell migration assay. PAE or PAE/KDR cells were seeded in the upper compartment, and were treated with the indicated concentrations of anosmin-1 or VEGF-A, or without anosmin-1 (Control). The cells that moved into the lower chamber were counted in five different microscopic fields. **P

    Techniques Used: Activation Assay, Migration, Expressing, Western Blot, Cell Migration Assay

    Angiogenic effects of anosmin-1 in endothelial cells. ( a ) Transwell cell migration assay. HUVECs or bEnd3 cells were seeded in the upper compartment, and were treated with the indicated concentrations of anosmin-1, VEGF-A, both 7.5 nM anosmin-1 and 1 nM VEGF-A (Anosmin-1 + VEGF-A), or without reagents (0 nM). The cells that migrated through the Transwell membrane and attached on the underside of the membrane were counted in five different microscopic fields and quantified. ( b ) Cell proliferation assay. Starved HUVECs or bEnd3 cells were treated with the indicated concentrations of anosmin-1, VEGF-A, both 7.5 nM anosmin-1 and 1 nM VEGF-A (Anosmin-1 + VEGF-A), or without reagents (0 nM). After the incubation, the number of cells in the culture dishes was counted. ( c ) Representative images of the 2D tube formation assay. Cells were seeded on a 24-well plate coated with Matrigel, and then, 7.5 nM anosmin-1, 1 nM VEGF-A or both 7.5 nM anosmin-1 and 1 nM VEGF-A (Anosmin-1 + VEGF-A) were added. As a negative control (Control), cells were incubated without reagents. After the incubation and fixation, the formed tubes were observed by light microscopy. Scale bar: 200 μm. ( d ) Summary graphs of ( c ). Total length of the formed tubes was measured. *
    Figure Legend Snippet: Angiogenic effects of anosmin-1 in endothelial cells. ( a ) Transwell cell migration assay. HUVECs or bEnd3 cells were seeded in the upper compartment, and were treated with the indicated concentrations of anosmin-1, VEGF-A, both 7.5 nM anosmin-1 and 1 nM VEGF-A (Anosmin-1 + VEGF-A), or without reagents (0 nM). The cells that migrated through the Transwell membrane and attached on the underside of the membrane were counted in five different microscopic fields and quantified. ( b ) Cell proliferation assay. Starved HUVECs or bEnd3 cells were treated with the indicated concentrations of anosmin-1, VEGF-A, both 7.5 nM anosmin-1 and 1 nM VEGF-A (Anosmin-1 + VEGF-A), or without reagents (0 nM). After the incubation, the number of cells in the culture dishes was counted. ( c ) Representative images of the 2D tube formation assay. Cells were seeded on a 24-well plate coated with Matrigel, and then, 7.5 nM anosmin-1, 1 nM VEGF-A or both 7.5 nM anosmin-1 and 1 nM VEGF-A (Anosmin-1 + VEGF-A) were added. As a negative control (Control), cells were incubated without reagents. After the incubation and fixation, the formed tubes were observed by light microscopy. Scale bar: 200 μm. ( d ) Summary graphs of ( c ). Total length of the formed tubes was measured. *

    Techniques Used: Cell Migration Assay, Proliferation Assay, Incubation, Tube Formation Assay, Negative Control, Light Microscopy

    Anosmin-1-induced activation of PKC and its involvement in tube formation. ( a ) Phosphorylation of PKC substrates by anosmin-1. After incubation with 10 nM siPLCγ1 or siControl for 2 days, starved HUVECs were treated with 7.5 nM anosmin-1, 1 nM VEGF-A, 10 nM PMA, or without reagents (Control) for 10 min. PMA was used as a positive control for the PKC activator. Cell lysates were analyzed by western blotting with the anti-phospho-PKC substrates and anti-β-actin Abs. Red asterisks indicate the bands for which the density was increased by anosmin-1 treatment, compared with those in the control lane. Blue asterisks indicate the bands for which the density was attenuated, compared with those of the siControl-treated samples. ( b–d ) Suppression of the anosmin-1-induced cell migration ( b ), proliferation (c) , and tube formation ( d ) by the PKC inhibitor. ( b ) HUVECs were seeded in the upper compartment and were treated with the indicated concentrations of anosmin-1, VEGF-A, or PMA in the presence of PKC inhibitor (10 μM GÖ6983) or 0.1% DMSO. The cells that moved into the lower chamber were counted in five different microscopic fields. ( c ) Starved HUVECs were treated with the indicated concentrations of anosmin-1, VEGF-A, or PMA in the presence of PKC inhibitor (10 μM GÖ6983) or 0.1% DMSO. After the incubation, the number of cells in the culture dishes was counted. (d) bEnd3 cells were seeded onto a Matrigel-coated 24-well plate, and were treated with the indicated concentrations of anosmin-1, VEGF-A, or PMA. At the same time, PKC inhibitor (10 μM GÖ6983) or 0.1% DMSO was added. Total length of the formed tubes was quantified and is summarized in the graph. *P
    Figure Legend Snippet: Anosmin-1-induced activation of PKC and its involvement in tube formation. ( a ) Phosphorylation of PKC substrates by anosmin-1. After incubation with 10 nM siPLCγ1 or siControl for 2 days, starved HUVECs were treated with 7.5 nM anosmin-1, 1 nM VEGF-A, 10 nM PMA, or without reagents (Control) for 10 min. PMA was used as a positive control for the PKC activator. Cell lysates were analyzed by western blotting with the anti-phospho-PKC substrates and anti-β-actin Abs. Red asterisks indicate the bands for which the density was increased by anosmin-1 treatment, compared with those in the control lane. Blue asterisks indicate the bands for which the density was attenuated, compared with those of the siControl-treated samples. ( b–d ) Suppression of the anosmin-1-induced cell migration ( b ), proliferation (c) , and tube formation ( d ) by the PKC inhibitor. ( b ) HUVECs were seeded in the upper compartment and were treated with the indicated concentrations of anosmin-1, VEGF-A, or PMA in the presence of PKC inhibitor (10 μM GÖ6983) or 0.1% DMSO. The cells that moved into the lower chamber were counted in five different microscopic fields. ( c ) Starved HUVECs were treated with the indicated concentrations of anosmin-1, VEGF-A, or PMA in the presence of PKC inhibitor (10 μM GÖ6983) or 0.1% DMSO. After the incubation, the number of cells in the culture dishes was counted. (d) bEnd3 cells were seeded onto a Matrigel-coated 24-well plate, and were treated with the indicated concentrations of anosmin-1, VEGF-A, or PMA. At the same time, PKC inhibitor (10 μM GÖ6983) or 0.1% DMSO was added. Total length of the formed tubes was quantified and is summarized in the graph. *P

    Techniques Used: Activation Assay, Incubation, Positive Control, Western Blot, Migration

    Anosmin-1-induced activation of VEGFR2 for tube formation. ( a ) Phosphorylation of VEGFR2 by anosmin-1 treatment. Starved HUVECs were stimulated with 7.5 nM anosmin-1 for the indicated durations or with 1 nM VEGF-A for 2 min. Cell lysates were analyzed by western blotting with the indicated Abs. The density of each phosphorylated VEGFR2 band normalized by the VEGFR2 band was measured, and the density relative to the value without treatment (0 min) set as 1.0 was calculated and is shown below the band. ( b ) Suppression of anosmin-1-induced tube formation by VEGFR2 inhibitor. HUVECs were seeded onto a Matrigel-coated 24-well plate, and then, 7.5 nM anosmin-1 or 1 nM VEGF-A was added with the VEGFR2 inhibitor (10 μM SU5614) or 0.1% DMSO at the same time. As a negative control (Control), cells were incubated without reagents in the presence of the VEGFR2 inhibitor or 0.1% DMSO. After the incubation and fixation, the formed tubes were observed by light microscopy. Scale bar: 500 μm. ( c ) Summary graphs of ( b ). Total length of the formed tubes was measured. **P
    Figure Legend Snippet: Anosmin-1-induced activation of VEGFR2 for tube formation. ( a ) Phosphorylation of VEGFR2 by anosmin-1 treatment. Starved HUVECs were stimulated with 7.5 nM anosmin-1 for the indicated durations or with 1 nM VEGF-A for 2 min. Cell lysates were analyzed by western blotting with the indicated Abs. The density of each phosphorylated VEGFR2 band normalized by the VEGFR2 band was measured, and the density relative to the value without treatment (0 min) set as 1.0 was calculated and is shown below the band. ( b ) Suppression of anosmin-1-induced tube formation by VEGFR2 inhibitor. HUVECs were seeded onto a Matrigel-coated 24-well plate, and then, 7.5 nM anosmin-1 or 1 nM VEGF-A was added with the VEGFR2 inhibitor (10 μM SU5614) or 0.1% DMSO at the same time. As a negative control (Control), cells were incubated without reagents in the presence of the VEGFR2 inhibitor or 0.1% DMSO. After the incubation and fixation, the formed tubes were observed by light microscopy. Scale bar: 500 μm. ( c ) Summary graphs of ( b ). Total length of the formed tubes was measured. **P

    Techniques Used: Activation Assay, Western Blot, Negative Control, Incubation, Light Microscopy

    BIAcore analysis for binding to VEGFR2. The data after the injection of VEGF-A ( a ), anosmin-1 WT ( b ), and anosmin-1 ∆2–4 (c) at the indicated concentrations are shown.
    Figure Legend Snippet: BIAcore analysis for binding to VEGFR2. The data after the injection of VEGF-A ( a ), anosmin-1 WT ( b ), and anosmin-1 ∆2–4 (c) at the indicated concentrations are shown.

    Techniques Used: Binding Assay, Injection

    Anosmin-1-induced activation of PLCγ1 and its involvement in tube formation. ( a,b ) Phosphorylation of PLCγ1 by anosmin-1. ( a ) Starved HUVECs were treated with the indicated concentration of anosmin-1 or VEGF-A, or without reagents (0 nM), for 5 min. ( b ) Starved bEnd3 cells were treated with 0.5 nM anosmin-1 or 1 nM VEGF-A for the indicated durations. After the treatment, cells were lysed and analyzed by western blotting with the anti-phospho-PLCγ1 and anti-PLCγ1 Abs. The density of each phosphorylated PLCγ1 band normalized by the PLCγ1 band was measured, and the density relative to the value without treatment (0 nM in a or 0 min in b ) set as 1.0 was calculated and is shown below the band. ( c ) Suppression of the anosmin-1-induced tube formation by the PLC inhibitor. HUVECs were seeded onto a Matrigel-coated 24-well plate and were treated with 7.5 nM anosmin-1 or 1 nM VEGF-A, or without reagents (Control). At the same time, PLC inhibitor (10 μM U73122) or 0.1% DMSO was added. After the incubation and fixation, the formed tubes were observed by light microscopy. Scale bar: 500 μm. ( d ) Summary graph of ( c ). Total length of the formed tubes was quantified. **P
    Figure Legend Snippet: Anosmin-1-induced activation of PLCγ1 and its involvement in tube formation. ( a,b ) Phosphorylation of PLCγ1 by anosmin-1. ( a ) Starved HUVECs were treated with the indicated concentration of anosmin-1 or VEGF-A, or without reagents (0 nM), for 5 min. ( b ) Starved bEnd3 cells were treated with 0.5 nM anosmin-1 or 1 nM VEGF-A for the indicated durations. After the treatment, cells were lysed and analyzed by western blotting with the anti-phospho-PLCγ1 and anti-PLCγ1 Abs. The density of each phosphorylated PLCγ1 band normalized by the PLCγ1 band was measured, and the density relative to the value without treatment (0 nM in a or 0 min in b ) set as 1.0 was calculated and is shown below the band. ( c ) Suppression of the anosmin-1-induced tube formation by the PLC inhibitor. HUVECs were seeded onto a Matrigel-coated 24-well plate and were treated with 7.5 nM anosmin-1 or 1 nM VEGF-A, or without reagents (Control). At the same time, PLC inhibitor (10 μM U73122) or 0.1% DMSO was added. After the incubation and fixation, the formed tubes were observed by light microscopy. Scale bar: 500 μm. ( d ) Summary graph of ( c ). Total length of the formed tubes was quantified. **P

    Techniques Used: Activation Assay, Concentration Assay, Western Blot, Planar Chromatography, Incubation, Light Microscopy

    11) Product Images from "VEGFA Upregulates FLJ10540 and Modulates Migration and Invasion of Lung Cancer via PI3K/AKT Pathway"

    Article Title: VEGFA Upregulates FLJ10540 and Modulates Migration and Invasion of Lung Cancer via PI3K/AKT Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005052

    FLJ10540 alone or increased FLJ10540, mediated by VEGF-A stimulation, not only modulates cell migration and invasion through the PI3K/AKT signaling pathway, but also reinforces PI3K complex formation. (A) Vehicle-CL 1-0 , CL 1-0 -FLJ10540, vehicle-RK3E, and RK3E-FLJ10540 infected cells were serum-starved for 24 hours and treated with or without the indicated inhibitors, including SB202190, PD98059, and LY294002 for 2 hours. The migration and invasion ratios of vehicle-CL 1-0 , CL 1-0 -HA-FLJ10540, vehicle-RK3E, and RK3E-Flag-FLJ10540 infected cells were determined as previously described. (B) CL 1-0 and RK3E-expressing FLJ10540 cells were treated with or without LY294002 at the final concentration of 10 µM. The total cell lysates were subjected to immunoblot analysis for the unphosphorylated or phosphorylated form of AKT. β-actin was used as an internal loading control. (C) CL 1-0 cells expressing HA-FLJ10540 were pre-treated with or without VEGF-A (20 ng/ml). After 10 min, LY294002 (10 µM) was added and cells were further incubated for 2 hours. The total cell lysates were subjected to immunoblot analysis for HA, FLJ10540, or the unphosphorylated or phosphorylated forms of AKT. (D) A negative control siRNA and two different FLJ10540 siRNAs (siFLJ10540-1 and siFLJ10540-2) were transfected into CL 1-0 cells for 24 hours. After transfection, cells were treated with or without VEGF-A (20 ng/ml) for 10 min. Western blotting was performed as in (C). (E upper panel) Serum-starved CL 1-0 cells were pre-treated with or without SU5416 for 2 hours; cells were then stimulated with or without 20 ng/ml VEGF-A for 10 min. Cell lysates were immunoprecipitated with polyclonal antibodies against p110-α or protein IgG (as a control), which was followed by immunoblotting with p110-α, p85-α, and FLJ10540. (E lower panel) A negative control siRNA and two different FLJ10540 siRNAs (siFLJ10540-1 and siFLJ10540-2) were transfected into CL 1-0 cells for 24 hours. After transfection, cell lysates were immunoprecipitated with polyclonal antibodies against p110-α, p85-α, or protein IgG (as a control), followed by immunoblotting with p110-α, p85-α, and FLJ10540.
    Figure Legend Snippet: FLJ10540 alone or increased FLJ10540, mediated by VEGF-A stimulation, not only modulates cell migration and invasion through the PI3K/AKT signaling pathway, but also reinforces PI3K complex formation. (A) Vehicle-CL 1-0 , CL 1-0 -FLJ10540, vehicle-RK3E, and RK3E-FLJ10540 infected cells were serum-starved for 24 hours and treated with or without the indicated inhibitors, including SB202190, PD98059, and LY294002 for 2 hours. The migration and invasion ratios of vehicle-CL 1-0 , CL 1-0 -HA-FLJ10540, vehicle-RK3E, and RK3E-Flag-FLJ10540 infected cells were determined as previously described. (B) CL 1-0 and RK3E-expressing FLJ10540 cells were treated with or without LY294002 at the final concentration of 10 µM. The total cell lysates were subjected to immunoblot analysis for the unphosphorylated or phosphorylated form of AKT. β-actin was used as an internal loading control. (C) CL 1-0 cells expressing HA-FLJ10540 were pre-treated with or without VEGF-A (20 ng/ml). After 10 min, LY294002 (10 µM) was added and cells were further incubated for 2 hours. The total cell lysates were subjected to immunoblot analysis for HA, FLJ10540, or the unphosphorylated or phosphorylated forms of AKT. (D) A negative control siRNA and two different FLJ10540 siRNAs (siFLJ10540-1 and siFLJ10540-2) were transfected into CL 1-0 cells for 24 hours. After transfection, cells were treated with or without VEGF-A (20 ng/ml) for 10 min. Western blotting was performed as in (C). (E upper panel) Serum-starved CL 1-0 cells were pre-treated with or without SU5416 for 2 hours; cells were then stimulated with or without 20 ng/ml VEGF-A for 10 min. Cell lysates were immunoprecipitated with polyclonal antibodies against p110-α or protein IgG (as a control), which was followed by immunoblotting with p110-α, p85-α, and FLJ10540. (E lower panel) A negative control siRNA and two different FLJ10540 siRNAs (siFLJ10540-1 and siFLJ10540-2) were transfected into CL 1-0 cells for 24 hours. After transfection, cell lysates were immunoprecipitated with polyclonal antibodies against p110-α, p85-α, or protein IgG (as a control), followed by immunoblotting with p110-α, p85-α, and FLJ10540.

    Techniques Used: Migration, Infection, Expressing, Concentration Assay, Incubation, Negative Control, Transfection, Western Blot, Immunoprecipitation

    Histopathologic and immunophenotypic findings in lung adenocarcinomas with immunoreactivity for VEGF-A, FLJ10540, and phosphorylated AKT. (A) The tumor has a papillary growth pattern (Hematoxylin Eosin, 100×). (B) The tumor cells are immunoreactive with VEGF-A, with a staining intensity score of 2 (100×). (C) The tumor cells are immunoreactive with FLJ10540, with a staining intensity score of 2 (100×). (D) The tumor cells are immunoreactive with phosphorylated AKT, with a staining intensity score of 3 (100×).
    Figure Legend Snippet: Histopathologic and immunophenotypic findings in lung adenocarcinomas with immunoreactivity for VEGF-A, FLJ10540, and phosphorylated AKT. (A) The tumor has a papillary growth pattern (Hematoxylin Eosin, 100×). (B) The tumor cells are immunoreactive with VEGF-A, with a staining intensity score of 2 (100×). (C) The tumor cells are immunoreactive with FLJ10540, with a staining intensity score of 2 (100×). (D) The tumor cells are immunoreactive with phosphorylated AKT, with a staining intensity score of 3 (100×).

    Techniques Used: Staining

    VEGF-A promotes FLJ10540 protein expression and enhances FLJ10540-induced migration and invasion in lung cancer cells. (A) The microarray expression patterns of VEGF-A and FLJ10540 in lung adenocarcinoma patients were shown. The results were normalized against the expression patterns of 56 chips (HG_U133A). N: adjacent non-tumor tissues; T: tumor tissues. (B, left panel) VEGF-A induced an increase in FLJ10540 protein levels in a dose-dependent manner. After treatment with various concentration of VEGF-A (left panel) or VEGF-B (right panel) for 10 min in CL 1-0 cells, the total proteins were extracted from CL 1-0 cells and probed with antibody against FLJ10540. (B, middle panel) Serum-starved CL 1-0 cells were pre-treated with or without various concentrations of SU5416 for 2 hours; cells were then stimulated with 20 ng/ml VEGF-A for 10 min. β-actin was used as an internal loading control. (C) Serum-starved CL 1-0 cells were pre-treated with or without SU5416 for 2 hours; cells were then stimulated with VEGF-A (at the concentration of 20 ng/ml) for 3 hours. The migration and invasion relative-folds were normalized against vehicle cells. (D, left, upper panel) For the migration assay, 5×10 3 cells of vehicle-CL 1-0 and CL 1-0 -FLJ10540 stable clones were seeded into the top of a Transwell insert and allowed to adhere for 12 hours, and were then incubated with or without VEGF-A (20 ng/ml) for 3 hours. At the end of the assay, the cells on the topside were scraped, and the cells that migrated to the bottom were fixed and stained with Giemsa. (D, left, bottom panel) For the invasion assay, 1×10 4 cells were seeded after Matrigel was added, and were then incubated with or without VEGF-A (20 ng/ml) for 3 hours. All of the data represent the mean±s.d. of three independent experiments. (E) Indirect immunofluorescence analysis of FLJ10540 in VEGF-A-treated cells. The protein expression and the subcellular localization of FLJ10540 were analyzed in the presence or absence of VEGF-A (20 ng/ml) in CL 1-0 cells using immunofluorescence microscopy. After being incubated with or without VEGF-A for 30 min or 180 min, the cells were fixed with 3.7% formaldehyde and processed for indirect immunofluorescence microscopy. FLJ10540 translocated to the cell membrane upon VEGF-A treatment (Arrowhead). Bar: 10 µm.
    Figure Legend Snippet: VEGF-A promotes FLJ10540 protein expression and enhances FLJ10540-induced migration and invasion in lung cancer cells. (A) The microarray expression patterns of VEGF-A and FLJ10540 in lung adenocarcinoma patients were shown. The results were normalized against the expression patterns of 56 chips (HG_U133A). N: adjacent non-tumor tissues; T: tumor tissues. (B, left panel) VEGF-A induced an increase in FLJ10540 protein levels in a dose-dependent manner. After treatment with various concentration of VEGF-A (left panel) or VEGF-B (right panel) for 10 min in CL 1-0 cells, the total proteins were extracted from CL 1-0 cells and probed with antibody against FLJ10540. (B, middle panel) Serum-starved CL 1-0 cells were pre-treated with or without various concentrations of SU5416 for 2 hours; cells were then stimulated with 20 ng/ml VEGF-A for 10 min. β-actin was used as an internal loading control. (C) Serum-starved CL 1-0 cells were pre-treated with or without SU5416 for 2 hours; cells were then stimulated with VEGF-A (at the concentration of 20 ng/ml) for 3 hours. The migration and invasion relative-folds were normalized against vehicle cells. (D, left, upper panel) For the migration assay, 5×10 3 cells of vehicle-CL 1-0 and CL 1-0 -FLJ10540 stable clones were seeded into the top of a Transwell insert and allowed to adhere for 12 hours, and were then incubated with or without VEGF-A (20 ng/ml) for 3 hours. At the end of the assay, the cells on the topside were scraped, and the cells that migrated to the bottom were fixed and stained with Giemsa. (D, left, bottom panel) For the invasion assay, 1×10 4 cells were seeded after Matrigel was added, and were then incubated with or without VEGF-A (20 ng/ml) for 3 hours. All of the data represent the mean±s.d. of three independent experiments. (E) Indirect immunofluorescence analysis of FLJ10540 in VEGF-A-treated cells. The protein expression and the subcellular localization of FLJ10540 were analyzed in the presence or absence of VEGF-A (20 ng/ml) in CL 1-0 cells using immunofluorescence microscopy. After being incubated with or without VEGF-A for 30 min or 180 min, the cells were fixed with 3.7% formaldehyde and processed for indirect immunofluorescence microscopy. FLJ10540 translocated to the cell membrane upon VEGF-A treatment (Arrowhead). Bar: 10 µm.

    Techniques Used: Expressing, Migration, Microarray, Concentration Assay, Clone Assay, Incubation, Staining, Invasion Assay, Immunofluorescence, Microscopy

    12) Product Images from "Histopathological investigation of glioblastomas resected under bevacizumab treatment"

    Article Title: Histopathological investigation of glioblastomas resected under bevacizumab treatment

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9387

    Immunohistochemical analysis for VEGF and VEGFRs in tumors resected under neoadjuvant bevacizumab (Bev) as compared with that in control glioblastomas A. Before Bev treatment in case 1, shows diffuse staining for VEGF. (VEGF-A, original magnification ×400. The magnification bar: 50 μm). B. After Bev treatment in case 1, shows diffuse staining for VEGF (VEGF-A, original magnification ×400. The magnification bar: 50 μm). C. After Bev treatment in case 2. The tumor is negative for VEGF. (VEGF-A, original magnification ×400. The magnification bar: 50 μm). D. Control glioblastoma, showing diffuse staining for VEGF. (VEGF-A, original magnification ×400. The magnification bar: 50 μm). E. After Bev treatment in case 3. The expression of VEGFR1 is not observed either in vascular endothelial cells or tumor cells. (VEGFR1, original magnification ×400. The magnification bar: 50 μm). F. Control glioblastoma, showing staining for VEGFR1 in vascular endothelial cells. (VEGFR1, original magnification ×400. The magnification bar: 50 μm.). G. After Bev treatment in case 3, shows faint staining for VEGFR2 in vascular endothelial cells. (VEGFR2, original magnification ×400. The magnification bar: 50 μm). H. Control glioblastoma, showing strong staining for VEGFR2 in vascular endothelial cells. (VEGFR2, original magnification ×400. The magnification bar: 50 μm).
    Figure Legend Snippet: Immunohistochemical analysis for VEGF and VEGFRs in tumors resected under neoadjuvant bevacizumab (Bev) as compared with that in control glioblastomas A. Before Bev treatment in case 1, shows diffuse staining for VEGF. (VEGF-A, original magnification ×400. The magnification bar: 50 μm). B. After Bev treatment in case 1, shows diffuse staining for VEGF (VEGF-A, original magnification ×400. The magnification bar: 50 μm). C. After Bev treatment in case 2. The tumor is negative for VEGF. (VEGF-A, original magnification ×400. The magnification bar: 50 μm). D. Control glioblastoma, showing diffuse staining for VEGF. (VEGF-A, original magnification ×400. The magnification bar: 50 μm). E. After Bev treatment in case 3. The expression of VEGFR1 is not observed either in vascular endothelial cells or tumor cells. (VEGFR1, original magnification ×400. The magnification bar: 50 μm). F. Control glioblastoma, showing staining for VEGFR1 in vascular endothelial cells. (VEGFR1, original magnification ×400. The magnification bar: 50 μm.). G. After Bev treatment in case 3, shows faint staining for VEGFR2 in vascular endothelial cells. (VEGFR2, original magnification ×400. The magnification bar: 50 μm). H. Control glioblastoma, showing strong staining for VEGFR2 in vascular endothelial cells. (VEGFR2, original magnification ×400. The magnification bar: 50 μm).

    Techniques Used: Immunohistochemistry, Staining, Expressing

    13) Product Images from "Primary Xenografts of Human Prostate Tissue as a Model to Study Angiogenesis Induced by Reactive Stroma"

    Article Title: Primary Xenografts of Human Prostate Tissue as a Model to Study Angiogenesis Induced by Reactive Stroma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029623

    The angiogenic burst in primary xenografts of prostate tissue is preceded by androgen-modulated up-regulation of VEGF-A gene expression in the stromal compartment. ( a ). PCR analysis of expression of transcripts for pro-angiogenic factors in initial prostate tissue specimens before transplantation, and in corresponding primary xenografts after transplantation. Total RNA was extracted from initial prostate tissue (IT), and from prostate xenografts on different days after transplantation (d1–d14). GADPH was used as an internal control. ( b ). Immuno-histochemical identification of human VEGF protein in primary xenografts of prostate tissue over the 14 days after transplantation (d1–d14) in host mice pre-implanted with (+T), or not pre-implanted with (−T), sustained-release testosterone pellets. Bars = 50 µm.
    Figure Legend Snippet: The angiogenic burst in primary xenografts of prostate tissue is preceded by androgen-modulated up-regulation of VEGF-A gene expression in the stromal compartment. ( a ). PCR analysis of expression of transcripts for pro-angiogenic factors in initial prostate tissue specimens before transplantation, and in corresponding primary xenografts after transplantation. Total RNA was extracted from initial prostate tissue (IT), and from prostate xenografts on different days after transplantation (d1–d14). GADPH was used as an internal control. ( b ). Immuno-histochemical identification of human VEGF protein in primary xenografts of prostate tissue over the 14 days after transplantation (d1–d14) in host mice pre-implanted with (+T), or not pre-implanted with (−T), sustained-release testosterone pellets. Bars = 50 µm.

    Techniques Used: Expressing, Polymerase Chain Reaction, Transplantation Assay, Mouse Assay

    Schematic representation of timeframe of the transplantation-induced biological processes in primary xenografts of human benign and malignant prostate tissue. ( a ) Temporal changes of VEGF-A expression (brown line), angiogenesis (blue line), microvessel density (red line) and expression of a reactive stroma phenotype after xenograft transplantation. ( b ) The data from graph (a) suggests two hypothetical models of the cause-effect relationship of VEGF-A expression with angiogenesis and reactive stroma generation in primary xenografts of human prostate tissue.
    Figure Legend Snippet: Schematic representation of timeframe of the transplantation-induced biological processes in primary xenografts of human benign and malignant prostate tissue. ( a ) Temporal changes of VEGF-A expression (brown line), angiogenesis (blue line), microvessel density (red line) and expression of a reactive stroma phenotype after xenograft transplantation. ( b ) The data from graph (a) suggests two hypothetical models of the cause-effect relationship of VEGF-A expression with angiogenesis and reactive stroma generation in primary xenografts of human prostate tissue.

    Techniques Used: Transplantation Assay, Expressing

    Induction of a reactive stroma in primary xenografts of human prostate tissue. Temporal changes of protein levels of VEGF, αSMA, Calponin and Vimentin were measured by IHC-staining, and of the presence of smooth muscle cells and collagen fibers was visualized by Masson's trichrome staining, over the 14 days following xenograft transplantation. α-SMA and Calponin are early and late markers of smooth muscle, respectively. Masson's trichrome identifies smooth muscle cells (purple) and collagen fibers (green).
    Figure Legend Snippet: Induction of a reactive stroma in primary xenografts of human prostate tissue. Temporal changes of protein levels of VEGF, αSMA, Calponin and Vimentin were measured by IHC-staining, and of the presence of smooth muscle cells and collagen fibers was visualized by Masson's trichrome staining, over the 14 days following xenograft transplantation. α-SMA and Calponin are early and late markers of smooth muscle, respectively. Masson's trichrome identifies smooth muscle cells (purple) and collagen fibers (green).

    Techniques Used: Immunohistochemistry, Staining, Transplantation Assay

    14) Product Images from "High Density Lipoprotein Nanoparticles Deliver RNAi to Endothelial Cells to Inhibit Angiogenesis"

    Article Title: High Density Lipoprotein Nanoparticles Deliver RNAi to Endothelial Cells to Inhibit Angiogenesis

    Journal: Particle & particle systems characterization : measurement and description of particle properties and behavior in powders and other disperse systems

    doi:

    Matrigel plug assay and in vivo function of RNAi-HDL NPs. Nude mice implanted with Matrigel plugs containing 400 ng ml −1 VEGF-A were systemically administered HDL NPs (HDL) or conjugates with RNAi (RNAi-HDL) and NS ONs (NS-HDL). In addition, free
    Figure Legend Snippet: Matrigel plug assay and in vivo function of RNAi-HDL NPs. Nude mice implanted with Matrigel plugs containing 400 ng ml −1 VEGF-A were systemically administered HDL NPs (HDL) or conjugates with RNAi (RNAi-HDL) and NS ONs (NS-HDL). In addition, free

    Techniques Used: Matrigel Assay, In Vivo, Mouse Assay

    RNAi-HDL NPs reduce VEGF-A responsive endothelial cell survival and morphogenesis. (a) Annexin V/PI flow-cytometric assay conducted on VEGF-A induced (grey, 20 ng/ml) or un-induced (black) HUVECs revealed that treatment with RNAi-HDL NPs (RNAi-HDL) leads
    Figure Legend Snippet: RNAi-HDL NPs reduce VEGF-A responsive endothelial cell survival and morphogenesis. (a) Annexin V/PI flow-cytometric assay conducted on VEGF-A induced (grey, 20 ng/ml) or un-induced (black) HUVECs revealed that treatment with RNAi-HDL NPs (RNAi-HDL) leads

    Techniques Used: Flow Cytometry

    15) Product Images from "Regulation of HIF-1? activity in adipose tissue by obesity-associated factors: adipogenesis, insulin, and hypoxia"

    Article Title: Regulation of HIF-1? activity in adipose tissue by obesity-associated factors: adipogenesis, insulin, and hypoxia

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    doi: 10.1152/ajpendo.00626.2010

    HIF-1α in transcriptional expression of VEGF. A : chromatin immunoprecipitation (ChIP) assay for HIF-1α in the VEGF gene promoter. The assay was conducted in differentiated 3T3-L1 adipocytes after hypoxia treatment for 8 h. B : hypoxia induction
    Figure Legend Snippet: HIF-1α in transcriptional expression of VEGF. A : chromatin immunoprecipitation (ChIP) assay for HIF-1α in the VEGF gene promoter. The assay was conducted in differentiated 3T3-L1 adipocytes after hypoxia treatment for 8 h. B : hypoxia induction

    Techniques Used: Expressing, Chromatin Immunoprecipitation

    HIF-1α regulation by cell differentiation. A : VEGF mRNA during adipogenesis of 3T3-L1. Total mRNA was prepared from cells collected at times as indicated and quantified for VEGF mRNA by qRT-PCR. B : HIF-1α protein in the whole cell lysate
    Figure Legend Snippet: HIF-1α regulation by cell differentiation. A : VEGF mRNA during adipogenesis of 3T3-L1. Total mRNA was prepared from cells collected at times as indicated and quantified for VEGF mRNA by qRT-PCR. B : HIF-1α protein in the whole cell lysate

    Techniques Used: Cell Differentiation, Quantitative RT-PCR

    Hypoxia-inducible factor-1 (HIF-1) function indicated by plasma vascular endothelial growth factor (VEGF) in obese mice. A : HIF-1α protein in the adipose tissue. Tissue homogenizer was made from epididymal fat of dietary obese mice and examined
    Figure Legend Snippet: Hypoxia-inducible factor-1 (HIF-1) function indicated by plasma vascular endothelial growth factor (VEGF) in obese mice. A : HIF-1α protein in the adipose tissue. Tissue homogenizer was made from epididymal fat of dietary obese mice and examined

    Techniques Used: Mouse Assay

    16) Product Images from "CLEC14A deficiency exacerbates neuronal loss by increasing blood-brain barrier permeability and inflammation"

    Article Title: CLEC14A deficiency exacerbates neuronal loss by increasing blood-brain barrier permeability and inflammation

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-020-1727-6

    CLEC14A knockdown decreased junctional integrity in ECs. a , d HUVEC and HBMEC monolayer permeability to FITC-dextran increased after knockdown of CLEC14A using siRNA(50 nM) and treatment with VEGF (50 ng/ml, 30 min). Absorbance of the solution in the lower chamber was measured after FITC-dextran was added to the transwell. b , e TEER was reduced by VEGF stimulation under the same conditions of the permeability assay before FITC-dextran was added. The TEER was measured using a Millicell ERS-2 (Millipore). c Immunofluorescence staining of VE-cadherin, ZO-1, and DAPI in untreated or VEGF-treated (50 ng/ml, 30 min) HUVECs after knockdown of CLEC14A. Each arrow indicates an attenuated junction. Scale bars: 20 μm. * P
    Figure Legend Snippet: CLEC14A knockdown decreased junctional integrity in ECs. a , d HUVEC and HBMEC monolayer permeability to FITC-dextran increased after knockdown of CLEC14A using siRNA(50 nM) and treatment with VEGF (50 ng/ml, 30 min). Absorbance of the solution in the lower chamber was measured after FITC-dextran was added to the transwell. b , e TEER was reduced by VEGF stimulation under the same conditions of the permeability assay before FITC-dextran was added. The TEER was measured using a Millicell ERS-2 (Millipore). c Immunofluorescence staining of VE-cadherin, ZO-1, and DAPI in untreated or VEGF-treated (50 ng/ml, 30 min) HUVECs after knockdown of CLEC14A. Each arrow indicates an attenuated junction. Scale bars: 20 μm. * P

    Techniques Used: Permeability, Immunofluorescence, Staining

    17) Product Images from "Human retinal pigment epithelial cell proliferation by the combined stimulation of hydroquinone and advanced glycation end-products via up-regulation of VEGF gene"

    Article Title: Human retinal pigment epithelial cell proliferation by the combined stimulation of hydroquinone and advanced glycation end-products via up-regulation of VEGF gene

    Journal: Biochemistry and Biophysics Reports

    doi: 10.1016/j.bbrep.2015.05.005

    Effect of SP1 knockdown on VEGF-A mRNA expression in ARPE-19 cells. After siRNA introduction, ARPE-19 cells were exposed with HQ+AGE. The mRNA levels of (A) SP1 and (B) VEGF-A were measured by real-time RT-PCR. Data are expressed as means±SEM for each group ( n =4).
    Figure Legend Snippet: Effect of SP1 knockdown on VEGF-A mRNA expression in ARPE-19 cells. After siRNA introduction, ARPE-19 cells were exposed with HQ+AGE. The mRNA levels of (A) SP1 and (B) VEGF-A were measured by real-time RT-PCR. Data are expressed as means±SEM for each group ( n =4).

    Techniques Used: Expressing, Quantitative RT-PCR

    Effects of site-directed mutations on VEGF-A promoter activity. (A) Three site-directed mutations from −102 to −83, VEGF M1–3 are indicated. VEGF M1 and M3 markedly decreased the promoter activity induced by HQ+AGEs. (B) Two site-directed mutations from −78 to −59, VEGF M4 and M5 are indicated. VEGF M4 markedly decreased the promoter activity. (C) Two site-directed mutations from −64 to −45, VEGF M6 and M7 are indicated. VEGF M6 markedly decreased the promoter activity. GC box sequences, which are possible binding sites for SP1 in the VEGF-A promoter, were shown by bold, and the mutation sites were underlined. Values are means±SEM for each group ( n =3).
    Figure Legend Snippet: Effects of site-directed mutations on VEGF-A promoter activity. (A) Three site-directed mutations from −102 to −83, VEGF M1–3 are indicated. VEGF M1 and M3 markedly decreased the promoter activity induced by HQ+AGEs. (B) Two site-directed mutations from −78 to −59, VEGF M4 and M5 are indicated. VEGF M4 markedly decreased the promoter activity. (C) Two site-directed mutations from −64 to −45, VEGF M6 and M7 are indicated. VEGF M6 markedly decreased the promoter activity. GC box sequences, which are possible binding sites for SP1 in the VEGF-A promoter, were shown by bold, and the mutation sites were underlined. Values are means±SEM for each group ( n =3).

    Techniques Used: Activity Assay, Binding Assay, Mutagenesis

    Inhibition of ARPE-19 cell proliferation by inhibition of VEGF signaling. (A) Effects of the VEGF inhibitors on cell proliferation. ARPE-19 were incubated with HQ+AGEs and three VEGF-A inhibitors, 10 µg/mL Sulochrin, 3 nM Ki8751 or 50 nM CBO-P11 for 12 h. After the treatment, cellular proliferation was measured by WST-8 assay. Data are exposed as means±SEM for each group ( n =6). (B) Effect of siRNA against VEGF-A on cell proliferation. SiRNA of VEGF-A was transfected into ARPE-19 cells and the cells were incubated with HQ+AGEs for 12 h. Cellular proliferation was measured by WST-8 assay. Data are expressed as means±SEM for each group ( n =5). (C) Effect of siRNA against RAGE on HQ+AGE-induced ARP19 cell proliferation. SiRNA of RAGE was transfected into ARPE-19 cells and the cells were incubated with HQ+AGEs for 12 h. Cellular proliferation was measured by WST-8 assay. Data are exposed as means±SEM for each group ( n =5).
    Figure Legend Snippet: Inhibition of ARPE-19 cell proliferation by inhibition of VEGF signaling. (A) Effects of the VEGF inhibitors on cell proliferation. ARPE-19 were incubated with HQ+AGEs and three VEGF-A inhibitors, 10 µg/mL Sulochrin, 3 nM Ki8751 or 50 nM CBO-P11 for 12 h. After the treatment, cellular proliferation was measured by WST-8 assay. Data are exposed as means±SEM for each group ( n =6). (B) Effect of siRNA against VEGF-A on cell proliferation. SiRNA of VEGF-A was transfected into ARPE-19 cells and the cells were incubated with HQ+AGEs for 12 h. Cellular proliferation was measured by WST-8 assay. Data are expressed as means±SEM for each group ( n =5). (C) Effect of siRNA against RAGE on HQ+AGE-induced ARP19 cell proliferation. SiRNA of RAGE was transfected into ARPE-19 cells and the cells were incubated with HQ+AGEs for 12 h. Cellular proliferation was measured by WST-8 assay. Data are exposed as means±SEM for each group ( n =5).

    Techniques Used: Inhibition, Incubation, Transfection

    Induction of VEGF-A expression by the addition of HQ and/or AGE. (A) Expression of VEGF-A mRNA in ARPE-19 cells. ARPE-19 cells were treated with no addition, HQ, AGEs or combinations for 12 h. The level of VEGF-A mRNA was measured by real-time RT-PCR using β-actin as an endogenous control. Data are expressed as means±SEM for each group ( n =4). (B) Concentrations of VEGF in the ARPE-19 culture medium were measured by ELISA. ARPE-19 cells were treated with no addition, HQ, AGEs or combinations for 12 h. Data are expressed as means±SEM for each group ( n =4).
    Figure Legend Snippet: Induction of VEGF-A expression by the addition of HQ and/or AGE. (A) Expression of VEGF-A mRNA in ARPE-19 cells. ARPE-19 cells were treated with no addition, HQ, AGEs or combinations for 12 h. The level of VEGF-A mRNA was measured by real-time RT-PCR using β-actin as an endogenous control. Data are expressed as means±SEM for each group ( n =4). (B) Concentrations of VEGF in the ARPE-19 culture medium were measured by ELISA. ARPE-19 cells were treated with no addition, HQ, AGEs or combinations for 12 h. Data are expressed as means±SEM for each group ( n =4).

    Techniques Used: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Localization of essential region for VEGF-A transcription. The promoter activity on deleted promoter of human VEGF-A gene was shown. A series of luciferase constructs containing promoter fragments with various 5ʹ-ends were transfected into (A) ARPE-19 and (B) h1RPE7 cells. The promoter activity was normalized and expressed relative to the activity of co-transfected β-galactosidase plasmid and was expressed relative to the activity of promoterless pGL4.17[ luc2 /neo]. Values are means±SEM for each group ( n =3–4). Possible binding sites for SP1 in the promoter region were black labeled in left panel.
    Figure Legend Snippet: Localization of essential region for VEGF-A transcription. The promoter activity on deleted promoter of human VEGF-A gene was shown. A series of luciferase constructs containing promoter fragments with various 5ʹ-ends were transfected into (A) ARPE-19 and (B) h1RPE7 cells. The promoter activity was normalized and expressed relative to the activity of co-transfected β-galactosidase plasmid and was expressed relative to the activity of promoterless pGL4.17[ luc2 /neo]. Values are means±SEM for each group ( n =3–4). Possible binding sites for SP1 in the promoter region were black labeled in left panel.

    Techniques Used: Activity Assay, Luciferase, Construct, Transfection, Plasmid Preparation, Binding Assay, Labeling

    18) Product Images from "A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in angiogenesis"

    Article Title: A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in angiogenesis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03278-w

    VEGFR regulates angiogenesis and tumor VM through YAP/TAZ in vitro. a Transient knockdown of YAP and/or TAZ in MCF10A overexpressing VEGFR2 decreases expression of ANG-2 and CYR61. Western blotting exposures indicate relative expression of YAP and TAZ. b , c VEGFR2 overexpression and VEGF treatment increases tube formation by MCF10A through YAP/TAZ. YAP and/or TAZ were transiently knocked down by siRNA in MCF10A stably overexpressing VEGFR2 and subjected to tube-formation assay on Matrigel 48 h after transfection alongside wild type MCF10A. For some conditions, cells were stimulated with 100 ng ml −1 VEGF or were treated with 100 nM verteporfin for the duration of the tube formation assay. Representative images are shown in b . Scale bar denotes 200 μm. Total tube formation was quantified in c ( n = 3). * p
    Figure Legend Snippet: VEGFR regulates angiogenesis and tumor VM through YAP/TAZ in vitro. a Transient knockdown of YAP and/or TAZ in MCF10A overexpressing VEGFR2 decreases expression of ANG-2 and CYR61. Western blotting exposures indicate relative expression of YAP and TAZ. b , c VEGFR2 overexpression and VEGF treatment increases tube formation by MCF10A through YAP/TAZ. YAP and/or TAZ were transiently knocked down by siRNA in MCF10A stably overexpressing VEGFR2 and subjected to tube-formation assay on Matrigel 48 h after transfection alongside wild type MCF10A. For some conditions, cells were stimulated with 100 ng ml −1 VEGF or were treated with 100 nM verteporfin for the duration of the tube formation assay. Representative images are shown in b . Scale bar denotes 200 μm. Total tube formation was quantified in c ( n = 3). * p

    Techniques Used: In Vitro, Expressing, Western Blot, Over Expression, Stable Transfection, Tube Formation Assay, Transfection

    YAP/TAZ are mediators of VEGF-induced angiogenesis ex vivo and in vivo. a – c Pharmacological inhibition of YAP/TAZ reduces angiogenesis ex vivo in a rat aorta model. Sections of aorta were cultured for 7 days in Matrigel with 100 ng ml −1 VEGF and the indicated concentrations of VP. Representative images are shown in a . Sprout area is quantified in b . Scale bar denotes 500 μm. c Immunostaining of aorta sections demonstrates that outgrowths are positive for VE-cadherin. Scale bar denotes 300 μm. Wimasis image analysis software was used to visualize sprouts ( n = 3). d Transient knockdown of YAP/TAZ or pharmacological inhibition of YAP/TAZ with VP reduces angiogenesis by HUVECs in vivo in Matrigel plug experiments. Five million cells were injected subcutaneously into mice with Matrigel and 200 ng mL −1 VEGF. VP was administered by intraperitoneal injection every other day. Plugs were excised after 1 week. Representative images are shown in the top two rows. In the bottom panels, angiogenesis in the Matrigel plugs was stained by IHC for the human endothelial cell marker hCD31. Scale bar denotes 500 μm. e YAP/TAZ inhibition reduces endogenous angiogenesis in vivo in Matrigel plug experiments. Matrigel plugs with 200 ng ml −1 VEGF were implanted subcutaneously in mice. VP was administered by intraperitoneal injection every other day. Plugs were excised after 2 weeks. Angiogenesis was assessed by IHC staining for mouse mCD31 endothelial cell marker. Scale bar denotes 500 μm. f – h YAP/TAZ inhibition diminishes angiogenesis in vivo in a mouse retinal model. Mice were injected with 1 mg kg −1 VEGF with or without 100 mg kg −1 VP at postnatal day 3 (P3) and 4 (P4). At P5, retinal blood vasculature was stained. Representative images of the retinal vessel density are shown in f , whereas g shows images of the vascular front. Scale bar denotes 100 μm f or 30 μm g . Number of filopodia (active angiogenesis) is quantified in h . Each data point represents the average of two retinas from a single mouse ( n = 4 for control, n = 3 for VP). * p
    Figure Legend Snippet: YAP/TAZ are mediators of VEGF-induced angiogenesis ex vivo and in vivo. a – c Pharmacological inhibition of YAP/TAZ reduces angiogenesis ex vivo in a rat aorta model. Sections of aorta were cultured for 7 days in Matrigel with 100 ng ml −1 VEGF and the indicated concentrations of VP. Representative images are shown in a . Sprout area is quantified in b . Scale bar denotes 500 μm. c Immunostaining of aorta sections demonstrates that outgrowths are positive for VE-cadherin. Scale bar denotes 300 μm. Wimasis image analysis software was used to visualize sprouts ( n = 3). d Transient knockdown of YAP/TAZ or pharmacological inhibition of YAP/TAZ with VP reduces angiogenesis by HUVECs in vivo in Matrigel plug experiments. Five million cells were injected subcutaneously into mice with Matrigel and 200 ng mL −1 VEGF. VP was administered by intraperitoneal injection every other day. Plugs were excised after 1 week. Representative images are shown in the top two rows. In the bottom panels, angiogenesis in the Matrigel plugs was stained by IHC for the human endothelial cell marker hCD31. Scale bar denotes 500 μm. e YAP/TAZ inhibition reduces endogenous angiogenesis in vivo in Matrigel plug experiments. Matrigel plugs with 200 ng ml −1 VEGF were implanted subcutaneously in mice. VP was administered by intraperitoneal injection every other day. Plugs were excised after 2 weeks. Angiogenesis was assessed by IHC staining for mouse mCD31 endothelial cell marker. Scale bar denotes 500 μm. f – h YAP/TAZ inhibition diminishes angiogenesis in vivo in a mouse retinal model. Mice were injected with 1 mg kg −1 VEGF with or without 100 mg kg −1 VP at postnatal day 3 (P3) and 4 (P4). At P5, retinal blood vasculature was stained. Representative images of the retinal vessel density are shown in f , whereas g shows images of the vascular front. Scale bar denotes 100 μm f or 30 μm g . Number of filopodia (active angiogenesis) is quantified in h . Each data point represents the average of two retinas from a single mouse ( n = 4 for control, n = 3 for VP). * p

    Techniques Used: Ex Vivo, In Vivo, Inhibition, Cell Culture, Immunostaining, Software, Injection, Mouse Assay, Staining, Immunohistochemistry, Marker

    Model for VEGFR and Hippo signaling in angiogenesis/VM. When VEGF binds to its receptor, VEGFR, signaling through PI3K and MAPK is initiated. This leads to the inhibition of MST/LATS and subsequent activation of YAP/TAZ. YAP and TAZ induce ANG-2 and CYR61 expression, leading to enhanced angiogenesis and vasculogenic mimicry in endothelial and tumor cell lines, respectively
    Figure Legend Snippet: Model for VEGFR and Hippo signaling in angiogenesis/VM. When VEGF binds to its receptor, VEGFR, signaling through PI3K and MAPK is initiated. This leads to the inhibition of MST/LATS and subsequent activation of YAP/TAZ. YAP and TAZ induce ANG-2 and CYR61 expression, leading to enhanced angiogenesis and vasculogenic mimicry in endothelial and tumor cell lines, respectively

    Techniques Used: Inhibition, Microscale Thermophoresis, Activation Assay, Expressing

    19) Product Images from "Association of angiopoietin-2, C-reactive protein and markers of obesity and insulin resistance with survival outcome in colorectal cancer"

    Article Title: Association of angiopoietin-2, C-reactive protein and markers of obesity and insulin resistance with survival outcome in colorectal cancer

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6606005

    Survival of colorectal cancer patients from surgery to death from any cause by Kaplan–Meier survival analysis. Survival between groups with high and low serum ( A ) CRP, ( B ) Ang-2 and ( C ) VEGF-A. ( D ) Survival of patients with AJCC stage II disease between groups with high and low serum CRP. Median values were used as cut points for high vs low values.
    Figure Legend Snippet: Survival of colorectal cancer patients from surgery to death from any cause by Kaplan–Meier survival analysis. Survival between groups with high and low serum ( A ) CRP, ( B ) Ang-2 and ( C ) VEGF-A. ( D ) Survival of patients with AJCC stage II disease between groups with high and low serum CRP. Median values were used as cut points for high vs low values.

    Techniques Used:

    20) Product Images from "Ca2+ Influx through Reverse Mode Na+/Ca2+ Exchange Is Critical for Vascular Endothelial Growth Factor-mediated Extracellular Signal-regulated Kinase (ERK) 1/2 Activation and Angiogenic Functions of Human Endothelial Cells *"

    Article Title: Ca2+ Influx through Reverse Mode Na+/Ca2+ Exchange Is Critical for Vascular Endothelial Growth Factor-mediated Extracellular Signal-regulated Kinase (ERK) 1/2 Activation and Angiogenic Functions of Human Endothelial Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.251777

    Effect of reverse mode NCX inhibitors on Ca 2+ transients. Representative time courses of Ca 2+ -sensitive Fluo-4NW fluorescence recorded during VEGF stimulation of HUVECs are shown. Serum-starved HUVECs were stimulated with VEGF (50 ng/ml) at time 0. A–C
    Figure Legend Snippet: Effect of reverse mode NCX inhibitors on Ca 2+ transients. Representative time courses of Ca 2+ -sensitive Fluo-4NW fluorescence recorded during VEGF stimulation of HUVECs are shown. Serum-starved HUVECs were stimulated with VEGF (50 ng/ml) at time 0. A–C

    Techniques Used: Fluorescence

    Effect of NCX siRNA on phospho-ERK1/2 and HUVEC proliferation. A , HUVECs transfected with control nontargeting siRNA or NCX1 targeting siRNA (100 n m for 48 h) were serum-starved and subsequently challenged with VEGF (50 ng/ml) for 10 min. ERK1/2 and PLCγ
    Figure Legend Snippet: Effect of NCX siRNA on phospho-ERK1/2 and HUVEC proliferation. A , HUVECs transfected with control nontargeting siRNA or NCX1 targeting siRNA (100 n m for 48 h) were serum-starved and subsequently challenged with VEGF (50 ng/ml) for 10 min. ERK1/2 and PLCγ

    Techniques Used: Transfection

    Proposed mechanism of reverse mode NCX involvement in VEGF-induced ERK1/2 phosphorylation. Bold lines show direct interactions. Dashed lines show indirect interactions. Activation of PLCγ downstream of VEGFR leads to the generation of diacylglycerol
    Figure Legend Snippet: Proposed mechanism of reverse mode NCX involvement in VEGF-induced ERK1/2 phosphorylation. Bold lines show direct interactions. Dashed lines show indirect interactions. Activation of PLCγ downstream of VEGFR leads to the generation of diacylglycerol

    Techniques Used: Activation Assay

    Effect of NCX inhibitors on angiogenesis functional assays. A , HUVECs were exposed to VEGF (50 ng/ml) for 48 h in the presence of the indicated concentrations of DCB and KBR-7943, in MCDB-131 containing 0.1%w/v BSA. Bar C , control; bar Am , amiloride.
    Figure Legend Snippet: Effect of NCX inhibitors on angiogenesis functional assays. A , HUVECs were exposed to VEGF (50 ng/ml) for 48 h in the presence of the indicated concentrations of DCB and KBR-7943, in MCDB-131 containing 0.1%w/v BSA. Bar C , control; bar Am , amiloride.

    Techniques Used: Functional Assay

    Effect of NCX inhibitors on VEGF-induced ERK1/2 phosphorylation. A , serum-starved HUVECs were preincubated (for 30 min) with 30 μ m DCB or vehicle (0.5% v/v Me 2 SO) prior to VEGF stimulation (50 ng/ml) for the times indicated. B , ERK1/2 and PLCγ
    Figure Legend Snippet: Effect of NCX inhibitors on VEGF-induced ERK1/2 phosphorylation. A , serum-starved HUVECs were preincubated (for 30 min) with 30 μ m DCB or vehicle (0.5% v/v Me 2 SO) prior to VEGF stimulation (50 ng/ml) for the times indicated. B , ERK1/2 and PLCγ

    Techniques Used:

    21) Product Images from "Spatial regulation of VEGF receptor endocytosis in angiogenesis"

    Article Title: Spatial regulation of VEGF receptor endocytosis in angiogenesis

    Journal: Nature cell biology

    doi: 10.1038/ncb2679

    Endothelial Dab2 and PAR-3 regulate angiogenic vessel growth a , Overview of the P6 control and Dab2 iΔEC retinal vasculature. Anti-Dab2 (white/red) staining is shown in top panels. ECs, Isolectin B4 (IB4). Bottom panels show higher magnification of the angiogenic front. b , Defects in the P6 Pard3 iΔEC retinal vasculature. Anti-PAR-3 and IB4 (green) staining in middle panels show successful deletion of PAR-3 in Pard3 iΔEC vessels. Residual round signals correspond to autofluorescent blood cells. Bottom panels show higher magnification of the angiogenic front. c, d , Quantitation of filopodia number and length, tip cell number, EC-covered area, EC proliferation and vessels branch points in Dab2 mutant (iΔEC) ( c ) or Pard3 mutant ( d ) retinas compared to the corresponding control littermates (Ctrl). Dab2 mutant n=7, Pard3 mutant n=3. Percentage of reduction is indicated. Data represent the means±s.d. P values, two-tailed Student’s t-test. e , Reduced uptake of labelled VEGF-A (red) at the Dab2 iΔEC or Pard3 iΔEC angiogenic front compared to control littermates. Green arrowheads indicate VEGF-A spots, white arrowheads marks ECs with no or little uptake. f , Statistical analysis of internalised Alexa-coupled VEGF-A in the angiogenic front and central plexus. Data represent the means±s.d. of 6 independent experiments. P values, two-tailed Student’s t-test.
    Figure Legend Snippet: Endothelial Dab2 and PAR-3 regulate angiogenic vessel growth a , Overview of the P6 control and Dab2 iΔEC retinal vasculature. Anti-Dab2 (white/red) staining is shown in top panels. ECs, Isolectin B4 (IB4). Bottom panels show higher magnification of the angiogenic front. b , Defects in the P6 Pard3 iΔEC retinal vasculature. Anti-PAR-3 and IB4 (green) staining in middle panels show successful deletion of PAR-3 in Pard3 iΔEC vessels. Residual round signals correspond to autofluorescent blood cells. Bottom panels show higher magnification of the angiogenic front. c, d , Quantitation of filopodia number and length, tip cell number, EC-covered area, EC proliferation and vessels branch points in Dab2 mutant (iΔEC) ( c ) or Pard3 mutant ( d ) retinas compared to the corresponding control littermates (Ctrl). Dab2 mutant n=7, Pard3 mutant n=3. Percentage of reduction is indicated. Data represent the means±s.d. P values, two-tailed Student’s t-test. e , Reduced uptake of labelled VEGF-A (red) at the Dab2 iΔEC or Pard3 iΔEC angiogenic front compared to control littermates. Green arrowheads indicate VEGF-A spots, white arrowheads marks ECs with no or little uptake. f , Statistical analysis of internalised Alexa-coupled VEGF-A in the angiogenic front and central plexus. Data represent the means±s.d. of 6 independent experiments. P values, two-tailed Student’s t-test.

    Techniques Used: Staining, Quantitation Assay, Mutagenesis, Two Tailed Test

    Increased VEGF uptake and sprouting in the Prkci iΔEC central retina a, b , VEGF-A (red, a ) or VEGF-C ( b ) uptake in the Prkci iΔEC central plexus. Green arrowheads indicate ligand spots. Cell nuclei, DAPI (blue); ECs, Isolectin B4 (IB4, white). c , Statistical analysis of internalised VEGFs as shown in ( a ) and ( b ). Data represent the means±s.d. of 6 independent experiments. P values, two-tailed Student’s t-test. d , Phenotype of the Isolectin B4-stained (IB4) P6 Prkci iΔEC retinal vasculature. Higher magnification of the angiogenic front (middle) and central plexus (bottom) are shown. e–g , Quantitation of vessels branch points, EC area and proliferation ( e , n=3), the number and length of filopodia and the number of distal sprout tips at the angiogenic front ( f , n=6), and the number of ectopic sprouts and filopodia ( g , n=6) in the central retina of Prkci mutants (iΔEC) compared to control (Ctrl) retinas. Data represent the means±s.d. P values, two-tailed Student’s t-test
    Figure Legend Snippet: Increased VEGF uptake and sprouting in the Prkci iΔEC central retina a, b , VEGF-A (red, a ) or VEGF-C ( b ) uptake in the Prkci iΔEC central plexus. Green arrowheads indicate ligand spots. Cell nuclei, DAPI (blue); ECs, Isolectin B4 (IB4, white). c , Statistical analysis of internalised VEGFs as shown in ( a ) and ( b ). Data represent the means±s.d. of 6 independent experiments. P values, two-tailed Student’s t-test. d , Phenotype of the Isolectin B4-stained (IB4) P6 Prkci iΔEC retinal vasculature. Higher magnification of the angiogenic front (middle) and central plexus (bottom) are shown. e–g , Quantitation of vessels branch points, EC area and proliferation ( e , n=3), the number and length of filopodia and the number of distal sprout tips at the angiogenic front ( f , n=6), and the number of ectopic sprouts and filopodia ( g , n=6) in the central retina of Prkci mutants (iΔEC) compared to control (Ctrl) retinas. Data represent the means±s.d. P values, two-tailed Student’s t-test

    Techniques Used: Two Tailed Test, Staining, Quantitation Assay

    Spatial differences in VEGF uptake in the retina a , Quantitation of Alexa-labelled VEGF-A in retinal ECs at 10 and 45 min after injection. The formation of intracellular spots was blocked by MiTMAB. Data represent the means±s.d. of 6 independent experiments. P values, ANOVA. b , VEGF-A uptake in blood vessels of Flk iΔEC mice. Green arrowheads indicate internalised Alexa dye-coupled VEGF-A (red). Cell nuclei, DAPI (blue); ECs, Isolectin B4 (IB4, white). Data represent the means±s.d. of 6 independent experiments. P values, two-tailed student t test. c , Spatial distribution of VEGF-A or VEGF-C (red) uptake in retinal vessels. Green arrowheads indicate internalised label spots. Cell nuclei, DAPI (blue); ECs, Isolectin B4 (IB4, white).
    Figure Legend Snippet: Spatial differences in VEGF uptake in the retina a , Quantitation of Alexa-labelled VEGF-A in retinal ECs at 10 and 45 min after injection. The formation of intracellular spots was blocked by MiTMAB. Data represent the means±s.d. of 6 independent experiments. P values, ANOVA. b , VEGF-A uptake in blood vessels of Flk iΔEC mice. Green arrowheads indicate internalised Alexa dye-coupled VEGF-A (red). Cell nuclei, DAPI (blue); ECs, Isolectin B4 (IB4, white). Data represent the means±s.d. of 6 independent experiments. P values, two-tailed student t test. c , Spatial distribution of VEGF-A or VEGF-C (red) uptake in retinal vessels. Green arrowheads indicate internalised label spots. Cell nuclei, DAPI (blue); ECs, Isolectin B4 (IB4, white).

    Techniques Used: Quantitation Assay, Injection, Mouse Assay, Two Tailed Test

    Dab2 and PAR-3 control VEGF receptor internalisation a , Alexa546-labelled VEGF-A or VEGF-C (red) accumulated in the perinuclear region of control mouse ECs at 30 min after stimulation, which was strongly reduced after knockdown of Dab2 or Pard3 . Actin, Phalloidin (green); nuclei, DAPI (blue). b, c , Quantitation of Alexa546-positive peri-nuclear VEGF-A ( b ) or VEGF-C ( c ) spots. Two different siRNAs were used for Dab2 and Pard3 in ( b ). Data represent the means±s.d. of 6 independent experiments. P values, two-tailed Student’s t-test. At least 100 cells were scored in each experiment. d, e , Biochemical detection of biotinylated (surface) VEGFR2 and VEGFR3 in stimulated control and Dab2 ( d ) or Pard3 ( e ) KD cells. Antibodies used for immunoblotting and molecular weight marker are indicated. f , Activation of Rac1 in control and Dab2 or Pard3 KD mouse ECs stimulated with VEGF-A or VEGF-C for 5 min, as indicated.
    Figure Legend Snippet: Dab2 and PAR-3 control VEGF receptor internalisation a , Alexa546-labelled VEGF-A or VEGF-C (red) accumulated in the perinuclear region of control mouse ECs at 30 min after stimulation, which was strongly reduced after knockdown of Dab2 or Pard3 . Actin, Phalloidin (green); nuclei, DAPI (blue). b, c , Quantitation of Alexa546-positive peri-nuclear VEGF-A ( b ) or VEGF-C ( c ) spots. Two different siRNAs were used for Dab2 and Pard3 in ( b ). Data represent the means±s.d. of 6 independent experiments. P values, two-tailed Student’s t-test. At least 100 cells were scored in each experiment. d, e , Biochemical detection of biotinylated (surface) VEGFR2 and VEGFR3 in stimulated control and Dab2 ( d ) or Pard3 ( e ) KD cells. Antibodies used for immunoblotting and molecular weight marker are indicated. f , Activation of Rac1 in control and Dab2 or Pard3 KD mouse ECs stimulated with VEGF-A or VEGF-C for 5 min, as indicated.

    Techniques Used: Quantitation Assay, Two Tailed Test, Molecular Weight, Marker, Activation Assay

    Negative regulation of VEGF receptor internalisation by aPKC a , Phosphorylation of a GST-Dab2 fusion protein by recombinant PKCλ reduced its interaction with VEGFR3 and VEGFR2 in pull down assays. Densiometric (DM) values are shown below bands. ATP for the kinase activation was added (+) or absent (−), as indicated. CBB, Coomassie Brilliant Blue staining of GST fusion proteins. b , Western blot showing reduced Dab2 phoshorylation at Serine 24 (pS24) in cultured ECs after 30 min incubation with 5µM aPKC inhibitor. c , Association of Dab2 with immunoprecipitated VEGFR3 (anti-R3) was enhanced in Prkci iΔEC lung lysate compared to control littermates. No specific bands were immunoprecipitated with IgG. VEGFR3-associated Dab2 lacked detectable phosphorylation in Ser24 (pS24). d , Effect of aPKC inhibition on VEGF-A- or VEGF-C-induced VEGF receptor internalisation at indicated time points. Cells were preincubated with 5 µM of aPKC inhibitor for 30 min. Surface VEGFR2 (R2) and VEGFR3 (R3) were identified by biotinylation. e , Increased activation of MAP kinase (p-ERK1/2) by VEGF-A or VEGF-C after aPKC inhibition in cultured mouse ECs. Effects on AKT phosphorylation were comparably modest. Total ERK1/2 and AKT are shown as loading controls. Molecular weight marker (kD) is indicated. f , Anti-aPKC (total), anti-phospho-aPKC (p-aPKC, Thr560) and Isolectin B4 (IB4) staining of the P6 control and Prkci iΔEC retinal vasculature. g , Anti-aPKC immunostaining (red) labels ECs (IB4, green) at the angiogenic front and in the central retinal plexus. h , Phospho-aPKC (p-aPKC, Thr560) immunosignals were weak at the angiogenic front in comparison to vessels of the central plexus. Higher magnification of p-aPKC signals and merged channels in insets is shown on the right.
    Figure Legend Snippet: Negative regulation of VEGF receptor internalisation by aPKC a , Phosphorylation of a GST-Dab2 fusion protein by recombinant PKCλ reduced its interaction with VEGFR3 and VEGFR2 in pull down assays. Densiometric (DM) values are shown below bands. ATP for the kinase activation was added (+) or absent (−), as indicated. CBB, Coomassie Brilliant Blue staining of GST fusion proteins. b , Western blot showing reduced Dab2 phoshorylation at Serine 24 (pS24) in cultured ECs after 30 min incubation with 5µM aPKC inhibitor. c , Association of Dab2 with immunoprecipitated VEGFR3 (anti-R3) was enhanced in Prkci iΔEC lung lysate compared to control littermates. No specific bands were immunoprecipitated with IgG. VEGFR3-associated Dab2 lacked detectable phosphorylation in Ser24 (pS24). d , Effect of aPKC inhibition on VEGF-A- or VEGF-C-induced VEGF receptor internalisation at indicated time points. Cells were preincubated with 5 µM of aPKC inhibitor for 30 min. Surface VEGFR2 (R2) and VEGFR3 (R3) were identified by biotinylation. e , Increased activation of MAP kinase (p-ERK1/2) by VEGF-A or VEGF-C after aPKC inhibition in cultured mouse ECs. Effects on AKT phosphorylation were comparably modest. Total ERK1/2 and AKT are shown as loading controls. Molecular weight marker (kD) is indicated. f , Anti-aPKC (total), anti-phospho-aPKC (p-aPKC, Thr560) and Isolectin B4 (IB4) staining of the P6 control and Prkci iΔEC retinal vasculature. g , Anti-aPKC immunostaining (red) labels ECs (IB4, green) at the angiogenic front and in the central retinal plexus. h , Phospho-aPKC (p-aPKC, Thr560) immunosignals were weak at the angiogenic front in comparison to vessels of the central plexus. Higher magnification of p-aPKC signals and merged channels in insets is shown on the right.

    Techniques Used: Recombinant, Activation Assay, Staining, Western Blot, Cell Culture, Incubation, Immunoprecipitation, Inhibition, Molecular Weight, Marker, Immunostaining

    22) Product Images from "Fibroblast Growth Factor-2 Autofeedback Regulation in Pituitary Folliculostellate TtT/GF Cells"

    Article Title: Fibroblast Growth Factor-2 Autofeedback Regulation in Pituitary Folliculostellate TtT/GF Cells

    Journal:

    doi: 10.1210/en.2008-1625

    Growth factor-mediated induction of TtT/GF cell proliferation. TtT/GF cells were serum starved for 24 h and subsequently treated with FGF-2 (30 ng/ml), VEGF-A (20 ng/ml), FGF-4 (20 ng/ml), HGF (10 ng/ml) or IGF-I (200 ng/ml) for 24 h (A) or FGF-2 (B)
    Figure Legend Snippet: Growth factor-mediated induction of TtT/GF cell proliferation. TtT/GF cells were serum starved for 24 h and subsequently treated with FGF-2 (30 ng/ml), VEGF-A (20 ng/ml), FGF-4 (20 ng/ml), HGF (10 ng/ml) or IGF-I (200 ng/ml) for 24 h (A) or FGF-2 (B)

    Techniques Used:

    23) Product Images from "DDA suppresses angiogenesis and tumor growth of colorectal cancer in vivo through decreasing VEGFR2 signaling"

    Article Title: DDA suppresses angiogenesis and tumor growth of colorectal cancer in vivo through decreasing VEGFR2 signaling

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11152

    DDA inhibited VEGFR2 signaling pathway in HUVECs ( A ) Cells were pretreated with indicated concentrations of DDA for 30 min, followed by the addition of VEGF-A (25 ng/ml) for another 30 min. Phosphorylation status of FAK, STAT3, Akt and ERK1/2 were then determined by immunoblotting. The compiled results of FAK ( B ), STAT3 ( C ), Akt ( D ) and ERK1/2 ( E ) phosphorylation are shown. Each column represents the mean ± S.E.M. of at least four independent experiments. * p
    Figure Legend Snippet: DDA inhibited VEGFR2 signaling pathway in HUVECs ( A ) Cells were pretreated with indicated concentrations of DDA for 30 min, followed by the addition of VEGF-A (25 ng/ml) for another 30 min. Phosphorylation status of FAK, STAT3, Akt and ERK1/2 were then determined by immunoblotting. The compiled results of FAK ( B ), STAT3 ( C ), Akt ( D ) and ERK1/2 ( E ) phosphorylation are shown. Each column represents the mean ± S.E.M. of at least four independent experiments. * p

    Techniques Used:

    DDA inhibited VEGF-A-induced tube formation of HUVECs and rat aorta ring microvessel sprouting ( A ) HUVECs were seeded on Matrigel in the presence of VEGF-A (25 ng/ml) with or without DDA at indicated concentrations. Cells were then photographed under phase-contrast after 16 h. (B) Rat aortic rings were placed in Matrigel and treated with indicated concentrations of DDA in the presence or absence of VEGF-A (25 ng/ml). The effects of DDA on microvessel sprouting were examined on day 8. Figures shown in (A) and ( B ) are representative of at least seven independent experiments with similar results. ( C ) Bar graphs show compiled data of average sprout arch numbers in (A) ( n = 8). * p
    Figure Legend Snippet: DDA inhibited VEGF-A-induced tube formation of HUVECs and rat aorta ring microvessel sprouting ( A ) HUVECs were seeded on Matrigel in the presence of VEGF-A (25 ng/ml) with or without DDA at indicated concentrations. Cells were then photographed under phase-contrast after 16 h. (B) Rat aortic rings were placed in Matrigel and treated with indicated concentrations of DDA in the presence or absence of VEGF-A (25 ng/ml). The effects of DDA on microvessel sprouting were examined on day 8. Figures shown in (A) and ( B ) are representative of at least seven independent experiments with similar results. ( C ) Bar graphs show compiled data of average sprout arch numbers in (A) ( n = 8). * p

    Techniques Used:

    DDA inhibited VEGF-A- or tumor-induced neovascularization ( A ) Matrigel containing VEGF-A and heparin was subcutaneously injected into C57BL/6 mice. Vehicle and DDA (2 or 5 mg/kg/day) was administered intraperitoneally. Hemoglobin levels in the Matrigel plug were quantified 7 days after implantation and shown in the lower panel of the chart. Each column represents the mean ± S.E.M. of six independent experiments. * p
    Figure Legend Snippet: DDA inhibited VEGF-A- or tumor-induced neovascularization ( A ) Matrigel containing VEGF-A and heparin was subcutaneously injected into C57BL/6 mice. Vehicle and DDA (2 or 5 mg/kg/day) was administered intraperitoneally. Hemoglobin levels in the Matrigel plug were quantified 7 days after implantation and shown in the lower panel of the chart. Each column represents the mean ± S.E.M. of six independent experiments. * p

    Techniques Used: Injection, Mouse Assay

    DDA inhibited VEGF-A-induced phosphorylation of VEGFR1 and VEGFR2 in HUVECs ( A ) Cells were pretreated with indicated concentrations of DDA for 30 min, followed by the addition of VEGF-A (25 ng/ml) for another 5 min. Phosphorylation status of VEGFR1 and VEGFR2 were then determined by immunoblotting. The compiled results of VEGFR1 ( B ) and VEGFR2 ( C ) are shown. Each column represents the mean ± S.E.M. of at least five independent experiments. * p
    Figure Legend Snippet: DDA inhibited VEGF-A-induced phosphorylation of VEGFR1 and VEGFR2 in HUVECs ( A ) Cells were pretreated with indicated concentrations of DDA for 30 min, followed by the addition of VEGF-A (25 ng/ml) for another 5 min. Phosphorylation status of VEGFR1 and VEGFR2 were then determined by immunoblotting. The compiled results of VEGFR1 ( B ) and VEGFR2 ( C ) are shown. Each column represents the mean ± S.E.M. of at least five independent experiments. * p

    Techniques Used:

    DDA disrupted VEGFR2 and NRP-1 interaction ( A ) Cells were pretreated with indicated concentrations of DDA for 30 min, followed by the addition of VEGF-A (25 ng/ml) for another 10 min. Cells were then harvested and immunoprecipitated with the NRP-1 antibody. The immunoprecipitated complex was then subjected to immunoblotting with an anti-VEGFR2 antibody. Typical bands representative of three independent experiments with similar results are shown. Immunoblotting confirming equal amount of immunoprecipitated VEGFR2 for each sample is shown at the bottom. IP, immunoprecipitation; IB, immunoblotting ( B ) HUVECs were transfected with negative control siRNA or NRP-1 siRNA for 48 h. After transfection, cells were starved for 16 h, and stimulated with VEGF-A (25 ng/ml) for another 24 h. Cell proliferation was determined as described in the “ Materials and Methods” section. Each column represents the mean ± S.E.M. of four independent experiments. * p
    Figure Legend Snippet: DDA disrupted VEGFR2 and NRP-1 interaction ( A ) Cells were pretreated with indicated concentrations of DDA for 30 min, followed by the addition of VEGF-A (25 ng/ml) for another 10 min. Cells were then harvested and immunoprecipitated with the NRP-1 antibody. The immunoprecipitated complex was then subjected to immunoblotting with an anti-VEGFR2 antibody. Typical bands representative of three independent experiments with similar results are shown. Immunoblotting confirming equal amount of immunoprecipitated VEGFR2 for each sample is shown at the bottom. IP, immunoprecipitation; IB, immunoblotting ( B ) HUVECs were transfected with negative control siRNA or NRP-1 siRNA for 48 h. After transfection, cells were starved for 16 h, and stimulated with VEGF-A (25 ng/ml) for another 24 h. Cell proliferation was determined as described in the “ Materials and Methods” section. Each column represents the mean ± S.E.M. of four independent experiments. * p

    Techniques Used: Immunoprecipitation, Transfection, Negative Control

    DDA inhibited VEGF-A-induced proliferation, migration and invasion of HUVECs HUVECs were starved in 2% FBS-containing M199 medium without ECGS for 16 h. After starvation, cells were pretreated with indicated concentrations of DDA followed by the stimulation with VEGF-A (25 ng/ml) for another 24 h. Cell viability ( A ) and cell proliferation ( B ) were then determined by MTT assay and BrdU incorporation assay. Each column represents the mean ± S.E.M. of at least three independent experiments performed in triplicate. * p
    Figure Legend Snippet: DDA inhibited VEGF-A-induced proliferation, migration and invasion of HUVECs HUVECs were starved in 2% FBS-containing M199 medium without ECGS for 16 h. After starvation, cells were pretreated with indicated concentrations of DDA followed by the stimulation with VEGF-A (25 ng/ml) for another 24 h. Cell viability ( A ) and cell proliferation ( B ) were then determined by MTT assay and BrdU incorporation assay. Each column represents the mean ± S.E.M. of at least three independent experiments performed in triplicate. * p

    Techniques Used: Migration, MTT Assay, BrdU Incorporation Assay

    24) Product Images from "Statins decrease vascular epithelial growth factor expression via down-regulation of receptor for advanced glycation end-products"

    Article Title: Statins decrease vascular epithelial growth factor expression via down-regulation of receptor for advanced glycation end-products

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2017.e00401

    Effect of siRNA against RAGE on HQ + AGE-induced VEGF expression. SiRNA of RAGE was transfected into h1RPE7 cells and the cells were incubated with HQ + AGE for 24 h. VEGF-A mRNA levels were measured by real-time RT-PCR using β-actin as an endogenous control. Data are expressed as mean ± SE for each group (n = 4).
    Figure Legend Snippet: Effect of siRNA against RAGE on HQ + AGE-induced VEGF expression. SiRNA of RAGE was transfected into h1RPE7 cells and the cells were incubated with HQ + AGE for 24 h. VEGF-A mRNA levels were measured by real-time RT-PCR using β-actin as an endogenous control. Data are expressed as mean ± SE for each group (n = 4).

    Techniques Used: Expressing, Transfection, Incubation, Quantitative RT-PCR

    VEGF concentrations in h1RPE7 culture medium. The h1RPE7 human RPE cells were treated with no addition (control), or no addition, lovastatin or atorvastatin in the presence of HQ + AGE for 24 h. Data are expressed as mean ± SE for each group (n = 4).
    Figure Legend Snippet: VEGF concentrations in h1RPE7 culture medium. The h1RPE7 human RPE cells were treated with no addition (control), or no addition, lovastatin or atorvastatin in the presence of HQ + AGE for 24 h. Data are expressed as mean ± SE for each group (n = 4).

    Techniques Used:

    Induction of VEGF-A expression by the addition of lovastatin or atorvastatin to HQ + AGE-damaged h1RPE7 cells. The h1RPE7 cells were treated with no addition (control), or no addition, lovastatin or atorvastatin in the presence of HQ + AGE for 24 h. VEGF-A mRNA was measured by real-time RT-PCR using β-actin as an endogenous control. Data are expressed as mean ± SE for each group (n = 4).
    Figure Legend Snippet: Induction of VEGF-A expression by the addition of lovastatin or atorvastatin to HQ + AGE-damaged h1RPE7 cells. The h1RPE7 cells were treated with no addition (control), or no addition, lovastatin or atorvastatin in the presence of HQ + AGE for 24 h. VEGF-A mRNA was measured by real-time RT-PCR using β-actin as an endogenous control. Data are expressed as mean ± SE for each group (n = 4).

    Techniques Used: Expressing, Quantitative RT-PCR

    Promoter analyses of RAGE (A) and VEGF-A (B). The activities of deleted promoters of the human RAGE and VEGF-A genes are shown. A series of luciferase constructs containing promoter fragments with various 5′-ends were transfected into h1RPE7 cells. After reporter plasmid(s) were introduced into h1RPE7 cells without HQ, AGE, nor statins, cells were incubated for 24 h, and medium was replaced with fresh medium containing atorvastatin or lovastatin with HQ + AGE and was then incubated for another 24 h. The promoter activity was normalized to the activity of co-transfected β-galactosidase plasmid and expressed relative to the activity of –766 without statin in RAGE promoter (A) and -2303 without stain in VEGF promoter (B). Values are mean ± SE for each group (n = 4). Statistical significance was expressed by A and B (vs –766 RAGE promoter without stain), and C and D (vs –253 RAGE promoter without stain).
    Figure Legend Snippet: Promoter analyses of RAGE (A) and VEGF-A (B). The activities of deleted promoters of the human RAGE and VEGF-A genes are shown. A series of luciferase constructs containing promoter fragments with various 5′-ends were transfected into h1RPE7 cells. After reporter plasmid(s) were introduced into h1RPE7 cells without HQ, AGE, nor statins, cells were incubated for 24 h, and medium was replaced with fresh medium containing atorvastatin or lovastatin with HQ + AGE and was then incubated for another 24 h. The promoter activity was normalized to the activity of co-transfected β-galactosidase plasmid and expressed relative to the activity of –766 without statin in RAGE promoter (A) and -2303 without stain in VEGF promoter (B). Values are mean ± SE for each group (n = 4). Statistical significance was expressed by A and B (vs –766 RAGE promoter without stain), and C and D (vs –253 RAGE promoter without stain).

    Techniques Used: Luciferase, Construct, Transfection, Plasmid Preparation, Incubation, Activity Assay, Staining

    25) Product Images from "VEGF neutralization plus CTLA-4 blockade alters soluble and cellular factors associated with enhancing lymphocyte infiltration and humoral recognition in melanoma"

    Article Title: VEGF neutralization plus CTLA-4 blockade alters soluble and cellular factors associated with enhancing lymphocyte infiltration and humoral recognition in melanoma

    Journal: Cancer immunology research

    doi: 10.1158/2326-6066.CIR-16-0084

    VEGF-A inhibits TNFα-mediated ICAM-1 and VCAM-1 expression and T-cell adhesion onto TEC. TEC were treated with VEGF-A, TNFα, and/or bevacizumab as indicated for 16 h. A. Immunoblot analysis of ICAM-1 and VCAM-1 expression in TEC. B. FACS
    Figure Legend Snippet: VEGF-A inhibits TNFα-mediated ICAM-1 and VCAM-1 expression and T-cell adhesion onto TEC. TEC were treated with VEGF-A, TNFα, and/or bevacizumab as indicated for 16 h. A. Immunoblot analysis of ICAM-1 and VCAM-1 expression in TEC. B. FACS

    Techniques Used: Expressing, FACS

    26) Product Images from "Isolation and Characterization of Circulating Lymphatic Endothelial Colony Forming Cells"

    Article Title: Isolation and Characterization of Circulating Lymphatic Endothelial Colony Forming Cells

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2015.11.015

    ECFC response to VEGF-A and VEGF-C
    Figure Legend Snippet: ECFC response to VEGF-A and VEGF-C

    Techniques Used:

    27) Product Images from "Astrocyte-derived VEGF-A drives blood-brain barrier disruption in CNS inflammatory disease"

    Article Title: Astrocyte-derived VEGF-A drives blood-brain barrier disruption in CNS inflammatory disease

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI60842

    Effects of VEGF-A on CLN-5 are mediated via eNOS.
    Figure Legend Snippet: Effects of VEGF-A on CLN-5 are mediated via eNOS.

    Techniques Used:

    GfapCre:Hif1a fl/fl mice show normal VEGF-A expression and BBB opening in inflammatory lesions.
    Figure Legend Snippet: GfapCre:Hif1a fl/fl mice show normal VEGF-A expression and BBB opening in inflammatory lesions.

    Techniques Used: Mouse Assay, Expressing

    Efficient inactivation of VEGF-A in the inflamed CNS in GfapCre:Vegfa fl/fl mice.
    Figure Legend Snippet: Efficient inactivation of VEGF-A in the inflamed CNS in GfapCre:Vegfa fl/fl mice.

    Techniques Used: Mouse Assay

    28) Product Images from "Angiogenic Functions of Voltage-gated Na+ Channels in Human Endothelial Cells: MODULATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) SIGNALING*"

    Article Title: Angiogenic Functions of Voltage-gated Na+ Channels in Human Endothelial Cells: MODULATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) SIGNALING*

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.187559

    A mechanistic model for the role of VGSC activity in VEGF HUVEC signaling. Heavy lines show direct interactions. Dashed lines show indirect interactions. Voltage-gated sodium channel (VGSC) activity is proposed to influence the [Ca 2+ ] i close to the plasma
    Figure Legend Snippet: A mechanistic model for the role of VGSC activity in VEGF HUVEC signaling. Heavy lines show direct interactions. Dashed lines show indirect interactions. Voltage-gated sodium channel (VGSC) activity is proposed to influence the [Ca 2+ ] i close to the plasma

    Techniques Used: Activity Assay

    TTX inhibits ERK1/2 activation upon VEGF stimulation of HUVEC. HUVECs were incubated with VEGF in the presence of TTX for the indicated times. A , Western blots of phospho-ERK1/2, total ERK1/2, and GAPDH ( n = 4). B , phospho-PLC γ 1, total PLCγ,
    Figure Legend Snippet: TTX inhibits ERK1/2 activation upon VEGF stimulation of HUVEC. HUVECs were incubated with VEGF in the presence of TTX for the indicated times. A , Western blots of phospho-ERK1/2, total ERK1/2, and GAPDH ( n = 4). B , phospho-PLC γ 1, total PLCγ,

    Techniques Used: Activation Assay, Incubation, Western Blot, Planar Chromatography

    Micromolar concentrations of TTX inhibit VEGF-induced B-Raf activation, PKCμ/PKD phosphorylation, and PKCα translocation to the membrane fraction. HUVECs were stimulated with VEGF with or without TTX. A , B-Raf was immunoprecipitated from
    Figure Legend Snippet: Micromolar concentrations of TTX inhibit VEGF-induced B-Raf activation, PKCμ/PKD phosphorylation, and PKCα translocation to the membrane fraction. HUVECs were stimulated with VEGF with or without TTX. A , B-Raf was immunoprecipitated from

    Techniques Used: Activation Assay, Translocation Assay, Immunoprecipitation

    Micromolar TTX enhances VEGF-induced Ca 2+ transients and abolishes VEGF-induced membrane depolarization. A , time courses of Ca 2+ -sensitive Fluo-4NW fluorescence during VEGF stimulation of HUVECs. HUVECs were stimulated with VEGF at time 0. Shown are unstimulated
    Figure Legend Snippet: Micromolar TTX enhances VEGF-induced Ca 2+ transients and abolishes VEGF-induced membrane depolarization. A , time courses of Ca 2+ -sensitive Fluo-4NW fluorescence during VEGF stimulation of HUVECs. HUVECs were stimulated with VEGF at time 0. Shown are unstimulated

    Techniques Used: Fluorescence

    VEGF activates ERK1/2 through a pathway that involves Src, PLCγ, PKC, and Ca 2+ . HUVECs were treated with 1 μ m Src inhibitor PP2, 1 μ m PLC inhibitor, 30 μ m PI3K inhibitor LY294002 ( A ); 1 μ m PKC inhibitor GF109203X,
    Figure Legend Snippet: VEGF activates ERK1/2 through a pathway that involves Src, PLCγ, PKC, and Ca 2+ . HUVECs were treated with 1 μ m Src inhibitor PP2, 1 μ m PLC inhibitor, 30 μ m PI3K inhibitor LY294002 ( A ); 1 μ m PKC inhibitor GF109203X,

    Techniques Used: Planar Chromatography

    29) Product Images from "Vascular Endothelial Growth Factor Directly Inhibits Primitive Neural Stem Cell Survival But Promotes Definitive Neural Stem Cell Survival"

    Article Title: Vascular Endothelial Growth Factor Directly Inhibits Primitive Neural Stem Cell Survival But Promotes Definitive Neural Stem Cell Survival

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0526-06.2006

    A , Adult brain tissue as well as d-NSC-derived clonal neurospheres from the adult brain express Flk1 mRNA. Clonal E14.5 brain neurospheres also expressed Flk1 mRNA. Nestin and control GAPDH mRNAs were expressed in adult brain tissue, clonal adult brain neurospheres, and clonal E14.5 brain neurospheres. B , Nestin and Flk1 mRNA expression were not changed by VEGF-A treatment of either p-NSCs or d-NSCs. Moreover, Brachyury mRNA expression was not induced by VEGF-A treatment of either p-NSCs or d-NSCs. C , Increasing concentrations of VEGF-A enhanced the formation of neurospheres from adult mouse d-NSCs. Moreover, this VEGF-A-induced increase in neurospheres was suppressed by the addition of SU1498, a VEGF-A signaling inhibitor. SU1498 also decreased the number of neurospheres in the absence of VEGF-A in the culture media. D , The enhanced numbers of E14.5 d-NSC spheres produced by VEGF-A were blocked by the addition of two VEGF-A inhibitors, VEGF-A antibodies and Flk1/Fc-soluble chimeric receptors. There was a significant interaction of VEGF-A dose and blocker ( F (3,20) = 36.86; p
    Figure Legend Snippet: A , Adult brain tissue as well as d-NSC-derived clonal neurospheres from the adult brain express Flk1 mRNA. Clonal E14.5 brain neurospheres also expressed Flk1 mRNA. Nestin and control GAPDH mRNAs were expressed in adult brain tissue, clonal adult brain neurospheres, and clonal E14.5 brain neurospheres. B , Nestin and Flk1 mRNA expression were not changed by VEGF-A treatment of either p-NSCs or d-NSCs. Moreover, Brachyury mRNA expression was not induced by VEGF-A treatment of either p-NSCs or d-NSCs. C , Increasing concentrations of VEGF-A enhanced the formation of neurospheres from adult mouse d-NSCs. Moreover, this VEGF-A-induced increase in neurospheres was suppressed by the addition of SU1498, a VEGF-A signaling inhibitor. SU1498 also decreased the number of neurospheres in the absence of VEGF-A in the culture media. D , The enhanced numbers of E14.5 d-NSC spheres produced by VEGF-A were blocked by the addition of two VEGF-A inhibitors, VEGF-A antibodies and Flk1/Fc-soluble chimeric receptors. There was a significant interaction of VEGF-A dose and blocker ( F (3,20) = 36.86; p

    Techniques Used: Derivative Assay, Expressing, Produced

    A , The number of wild-type Flk1 +/+ p-NSC spheres is decreased by the addition of 100 ng/ml VEGF-A into the ES culture minimal medium ( t (4) = 2.6; p
    Figure Legend Snippet: A , The number of wild-type Flk1 +/+ p-NSC spheres is decreased by the addition of 100 ng/ml VEGF-A into the ES culture minimal medium ( t (4) = 2.6; p

    Techniques Used:

    30) Product Images from "HIF-1α-PDK1 axis-induced active glycolysis plays an essential role in macrophage migratory capacity"

    Article Title: HIF-1α-PDK1 axis-induced active glycolysis plays an essential role in macrophage migratory capacity

    Journal: Nature Communications

    doi: 10.1038/ncomms11635

    The role of glycolytic reprogramming in macrophage migratory capacity in vitro . ( a ) (i-iii) Macrophage migration activity to various chemoattractants (the supernatant of necrotic cell debris (i), stromal cell-derived factor 1 (SDF-1; 100 ng ml −1 ; ii) and VEGF-A (100 ng ml −1 ; iii)) was measured by transwell assay in normoxia (21% O 2 , 4 h), mild hypoxia (4% O 2 , 4 h) and severe hypoxia (1% O 2 , 4 h). The culture mediums were supplemented with 10 mM glucose (Glu) only, 2 mM L-glutamine (Gln) only or both. The y axis represents the migration activity relative to untreated condition. n =3 animals. ( b ) Macrophage migration activity was measured in mild hypoxia in the presence of 2-DG (20 mM). n =3 animals. ( c ) Migration activity in wild-type and HIF-1α-deficient macrophages was measured in the same manner as 4 ( a ). n =3 animals per group. ( d ) Macrophage migration activity to the supernatant of necrotic cell debris was measured in mild hypoxia in the presence of DCA (10 mM). n =3 animals per group. ( e ) (i) The number of filopodia in TEPMs is shown. TEPMs were cultured under mild hypoxia in the presence or absence of DCA (10 mM). (average with s.d. n =100 cells analysed). (ii) Representative immunocytochemical staining of Hoechst (blue), PKM2 (green) and Phalloidin (magenta) in TEPMs under mild hypoxia is shown. F-actin and PKM2 were co-localized in lamellipodia (magnified view 1, dotted line) and filopodia (magnified view 2, arrowheads). Scale bars, 10 μm. All graphs indicate average with s.d.. Student's t -test was performed to calculate P value. * P
    Figure Legend Snippet: The role of glycolytic reprogramming in macrophage migratory capacity in vitro . ( a ) (i-iii) Macrophage migration activity to various chemoattractants (the supernatant of necrotic cell debris (i), stromal cell-derived factor 1 (SDF-1; 100 ng ml −1 ; ii) and VEGF-A (100 ng ml −1 ; iii)) was measured by transwell assay in normoxia (21% O 2 , 4 h), mild hypoxia (4% O 2 , 4 h) and severe hypoxia (1% O 2 , 4 h). The culture mediums were supplemented with 10 mM glucose (Glu) only, 2 mM L-glutamine (Gln) only or both. The y axis represents the migration activity relative to untreated condition. n =3 animals. ( b ) Macrophage migration activity was measured in mild hypoxia in the presence of 2-DG (20 mM). n =3 animals. ( c ) Migration activity in wild-type and HIF-1α-deficient macrophages was measured in the same manner as 4 ( a ). n =3 animals per group. ( d ) Macrophage migration activity to the supernatant of necrotic cell debris was measured in mild hypoxia in the presence of DCA (10 mM). n =3 animals per group. ( e ) (i) The number of filopodia in TEPMs is shown. TEPMs were cultured under mild hypoxia in the presence or absence of DCA (10 mM). (average with s.d. n =100 cells analysed). (ii) Representative immunocytochemical staining of Hoechst (blue), PKM2 (green) and Phalloidin (magenta) in TEPMs under mild hypoxia is shown. F-actin and PKM2 were co-localized in lamellipodia (magnified view 1, dotted line) and filopodia (magnified view 2, arrowheads). Scale bars, 10 μm. All graphs indicate average with s.d.. Student's t -test was performed to calculate P value. * P

    Techniques Used: In Vitro, Migration, Activity Assay, Derivative Assay, Transwell Assay, Cell Culture, Staining

    31) Product Images from "HSV-1 Targets Lymphatic Vessels in the Eye and Draining Lymph Node of Mice Leading to Edema in the Absence of a Functional Type I Interferon Response"

    Article Title: HSV-1 Targets Lymphatic Vessels in the Eye and Draining Lymph Node of Mice Leading to Edema in the Absence of a Functional Type I Interferon Response

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2013.06.014

    HSV-1 infected CD118 − / − mice are not deficient in VEGF-A or VEGF receptor 2 expression. A : Cornea levels of VEGF-A, VEGF-C, and VEGF-D were quantified by real-time RT-PCR and normalized to the housekeeping gene β-actin on day 5
    Figure Legend Snippet: HSV-1 infected CD118 − / − mice are not deficient in VEGF-A or VEGF receptor 2 expression. A : Cornea levels of VEGF-A, VEGF-C, and VEGF-D were quantified by real-time RT-PCR and normalized to the housekeeping gene β-actin on day 5

    Techniques Used: Infection, Mouse Assay, Expressing, Quantitative RT-PCR

    32) Product Images from "Annexin II regulates fibrin homeostasis and neoangiogenesis in vivo"

    Article Title: Annexin II regulates fibrin homeostasis and neoangiogenesis in vivo

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI200419684

    Neovascularization of Matrigel plugs. ( a ) Annexin II immunostaining. Matrigel plugs containing VEGF-A (2 μg/ml) were implanted in wild-type mice, harvested on day 12, and stained with no primary antibody (Con), anti-annexin II (A II) IgG, or anti-annexin IV (A IV) IgG. Scale bar indicates 0.05 mm. ( b ) Neovascularization in annexin II–deficient mice. Matrigel plugs containing VEGF-A (100 ng/ml) or bFGF (100 ng/ml) were implanted in wild-type and annexin II–deficient mice, harvested on day 12, fixed, sectioned, and stained with H E. Scale bar indicates 0.05 mm. ( c ) Effect of annexin II N-terminal peptide. Matrigel plugs contained either sense peptide (STVHEILCKLSL; 250 μM), mimicking sequences required for t-PA binding to annexin II, or a scrambled control peptide (SLTSVLHKECLI; 250 μM) were analyzed on day 12 by enumeration of cells in sections stained with H E (10 random fields per section, 10 sections per sample, four mice per condition). Shown are mean cells per 200× field (SE; n = 4 mice). ( d ) Histological analysis of Matrigel plugs containing scrambled or sense tail peptide (H E stain). Scale bar indicates 0.05 mm.
    Figure Legend Snippet: Neovascularization of Matrigel plugs. ( a ) Annexin II immunostaining. Matrigel plugs containing VEGF-A (2 μg/ml) were implanted in wild-type mice, harvested on day 12, and stained with no primary antibody (Con), anti-annexin II (A II) IgG, or anti-annexin IV (A IV) IgG. Scale bar indicates 0.05 mm. ( b ) Neovascularization in annexin II–deficient mice. Matrigel plugs containing VEGF-A (100 ng/ml) or bFGF (100 ng/ml) were implanted in wild-type and annexin II–deficient mice, harvested on day 12, fixed, sectioned, and stained with H E. Scale bar indicates 0.05 mm. ( c ) Effect of annexin II N-terminal peptide. Matrigel plugs contained either sense peptide (STVHEILCKLSL; 250 μM), mimicking sequences required for t-PA binding to annexin II, or a scrambled control peptide (SLTSVLHKECLI; 250 μM) were analyzed on day 12 by enumeration of cells in sections stained with H E (10 random fields per section, 10 sections per sample, four mice per condition). Shown are mean cells per 200× field (SE; n = 4 mice). ( d ) Histological analysis of Matrigel plugs containing scrambled or sense tail peptide (H E stain). Scale bar indicates 0.05 mm.

    Techniques Used: Immunostaining, Mouse Assay, Staining, Binding Assay, H&E Stain

    Endothelial cell function. ( a ) VEGF-A-directed migration of wild-type (white bars) and annexin II–deficient (black bars) microvascular endothelial cells across collagen I and fibrin barriers. Shown are mean ± SE, n = 5 experiments. ( b ) Thoracic aortic rings embedded in type I collagen gels and incubated in serum-containing medium for 6 days. Shown are phase-contrast images of branching vessel-like structures in wild-type and annexin II–deficient explants. Scale bar, 0.25 mm. ( c ) t-PA-dependent plasmin generation in the presence of wild-type (filled circles) versus annexin II–null (open circles) microvascular endothelial cells expressed as relative fluorescence units (RFU). ( d ) MMP-9 activity in medium conditioned by aortic ring explants from wild-type (white bars) and annexin II–null (black bars) mice in the absence (–PLG) and presence (+PLG) of added plasminogen (2 μg/ml). Shown are mean ± SE, n = 6 experiments. ( e ) Media from annexin II–deficient (KO) and wild-type aortic ring explants obtained after culture, analyzed by Western blot using monoclonal anti–MMP-13 IgG and chemiluminescence. The putative active MMP-13 fragment migrates at about 48 kDa and proMMP-13, at about 60 kDa. ( f ) Densitometric analysis of MMP-13 Western blots of medium from cultures of wild-type (white bars) and annexin II–deficient (black bars) aortic rings. Shown are mean ± SE, n = 5 experiments.
    Figure Legend Snippet: Endothelial cell function. ( a ) VEGF-A-directed migration of wild-type (white bars) and annexin II–deficient (black bars) microvascular endothelial cells across collagen I and fibrin barriers. Shown are mean ± SE, n = 5 experiments. ( b ) Thoracic aortic rings embedded in type I collagen gels and incubated in serum-containing medium for 6 days. Shown are phase-contrast images of branching vessel-like structures in wild-type and annexin II–deficient explants. Scale bar, 0.25 mm. ( c ) t-PA-dependent plasmin generation in the presence of wild-type (filled circles) versus annexin II–null (open circles) microvascular endothelial cells expressed as relative fluorescence units (RFU). ( d ) MMP-9 activity in medium conditioned by aortic ring explants from wild-type (white bars) and annexin II–null (black bars) mice in the absence (–PLG) and presence (+PLG) of added plasminogen (2 μg/ml). Shown are mean ± SE, n = 6 experiments. ( e ) Media from annexin II–deficient (KO) and wild-type aortic ring explants obtained after culture, analyzed by Western blot using monoclonal anti–MMP-13 IgG and chemiluminescence. The putative active MMP-13 fragment migrates at about 48 kDa and proMMP-13, at about 60 kDa. ( f ) Densitometric analysis of MMP-13 Western blots of medium from cultures of wild-type (white bars) and annexin II–deficient (black bars) aortic rings. Shown are mean ± SE, n = 5 experiments.

    Techniques Used: Cell Function Assay, Migration, Incubation, Fluorescence, Activity Assay, Mouse Assay, Western Blot

    33) Product Images from "Pattern of expression of vascular endothelial growth factor and its receptors in the ovine choroid plexus during long and short photoperiods"

    Article Title: Pattern of expression of vascular endothelial growth factor and its receptors in the ovine choroid plexus during long and short photoperiods

    Journal: Cell and Tissue Research

    doi: 10.1007/s00441-012-1431-7

    Representative photographs of RT-PCR products. Two isoforms of VEGF-A ( line 1 ) were found in choroid plexus samples, VEGF-A 164 (275 bp) and VEGF-A 120 (125 bp). Single transcripts for Flt-1 (68 bp) ( line 2 ), KDR (145 bp) ( line 3 ) and NRP-1 (60 bp) ( line 4 ) were observed
    Figure Legend Snippet: Representative photographs of RT-PCR products. Two isoforms of VEGF-A ( line 1 ) were found in choroid plexus samples, VEGF-A 164 (275 bp) and VEGF-A 120 (125 bp). Single transcripts for Flt-1 (68 bp) ( line 2 ), KDR (145 bp) ( line 3 ) and NRP-1 (60 bp) ( line 4 ) were observed

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    Specificity of the VEGF-A ( a ), Flt-1 ( b ) and KDR ( c ) antibodies in western blot analyses of ovine choroid plexus (CP) homogenates. Samples were probed with either free antibody or antibody pre-adsorbed with specific control peptides. d Representative blots of recombinant human (rh)VEGF-A 121 ( line 1 ), ovine CPs ( lines 2 and 3 ) and rhVEGF-A 165 ( lines 4 and 5 ) resolved by SDS-PAGE and immunoblotted with VEGF-A antibodies used in ( a ). Blots in ( a ) were re-probed with β-actin antibodies. A antibodies, PA pre-adsorbed antibodies, NSB non-specific binding
    Figure Legend Snippet: Specificity of the VEGF-A ( a ), Flt-1 ( b ) and KDR ( c ) antibodies in western blot analyses of ovine choroid plexus (CP) homogenates. Samples were probed with either free antibody or antibody pre-adsorbed with specific control peptides. d Representative blots of recombinant human (rh)VEGF-A 121 ( line 1 ), ovine CPs ( lines 2 and 3 ) and rhVEGF-A 165 ( lines 4 and 5 ) resolved by SDS-PAGE and immunoblotted with VEGF-A antibodies used in ( a ). Blots in ( a ) were re-probed with β-actin antibodies. A antibodies, PA pre-adsorbed antibodies, NSB non-specific binding

    Techniques Used: Western Blot, Recombinant, SDS Page, Binding Assay

    Expression of VEGF-A 120 and VEGF-A 164 isoforms ( a ), Flt-1 ( b ), KDR ( c ) and NRP-1 ( d ) mRNA levels in the ovine choroid plexuses during short days (SD; 8L:16D) and long days (LD; 16L:8D). All values are presented as the mean ± SEM of ratios relative to PPIC determined by real-time PCR ( n = 5 /SD or LD photoperiod). * p
    Figure Legend Snippet: Expression of VEGF-A 120 and VEGF-A 164 isoforms ( a ), Flt-1 ( b ), KDR ( c ) and NRP-1 ( d ) mRNA levels in the ovine choroid plexuses during short days (SD; 8L:16D) and long days (LD; 16L:8D). All values are presented as the mean ± SEM of ratios relative to PPIC determined by real-time PCR ( n = 5 /SD or LD photoperiod). * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Distribution of vascular endothelial growth factor (VEGF-A) in the ovine choroid plexus. The red immunoreactivity of VEGF-A is strongly expressed in cobblestone-shaped epithelial cells ( arrow ), in comparison with the weaker immunoreactive signal observed in the spindle-shaped endothelial cells ( arrowheads ). Magnification ×200
    Figure Legend Snippet: Distribution of vascular endothelial growth factor (VEGF-A) in the ovine choroid plexus. The red immunoreactivity of VEGF-A is strongly expressed in cobblestone-shaped epithelial cells ( arrow ), in comparison with the weaker immunoreactive signal observed in the spindle-shaped endothelial cells ( arrowheads ). Magnification ×200

    Techniques Used:

    Western blot analyses of VEGF-A: VEGF-A 164 dimer ( a ), VEGF-A 164 monomers 21 and 23 kDa ( b ), Flt-1 ( c ) and KDR ( d ) in ovine choroid plexuses (CPs) during short days (SD; 8L:16D) and long days (LD; 16L:8D). Upper panels representative blots of CPs resolved by SDS-PAGE and immunoblotted with VEGF-A, Flt-1 and KDR antibodies. VEGF-A was visualized with alkaline phosphatase, whereas Flt-1 and KDR were visualized using the WesternDot™ 625 Western Blot Kit, which requires UV light for visualization. Lower panels mean ± SEM of the densitometric analysis of relative protein levels. * p
    Figure Legend Snippet: Western blot analyses of VEGF-A: VEGF-A 164 dimer ( a ), VEGF-A 164 monomers 21 and 23 kDa ( b ), Flt-1 ( c ) and KDR ( d ) in ovine choroid plexuses (CPs) during short days (SD; 8L:16D) and long days (LD; 16L:8D). Upper panels representative blots of CPs resolved by SDS-PAGE and immunoblotted with VEGF-A, Flt-1 and KDR antibodies. VEGF-A was visualized with alkaline phosphatase, whereas Flt-1 and KDR were visualized using the WesternDot™ 625 Western Blot Kit, which requires UV light for visualization. Lower panels mean ± SEM of the densitometric analysis of relative protein levels. * p

    Techniques Used: Western Blot, SDS Page

    34) Product Images from "Melatonin Decreases Pulmonary Vascular Remodeling and Oxygen Sensitivity in Pulmonary Hypertensive Newborn Lambs"

    Article Title: Melatonin Decreases Pulmonary Vascular Remodeling and Oxygen Sensitivity in Pulmonary Hypertensive Newborn Lambs

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2018.00185

    Immunolocalization and protein expression of VEGF and HIF in lung tissue. Representative micrographs (40x) showing immunohistochemical distribution of VEGF-A in vascular wall of lung resistance arteries (A) , and analysis for VEGF-A vascular immunoreactivity intensity (B) . Scale bar: 100 μm. Scanned photograph of immunoblots (C) , and HIF-1α (D) and VEGF (E) protein expression. Protein expression was referred to β-actin as control protein. Groups are control (CN, open bars, n = 5) and melatonin treated (MN, closed bars, n = 5) lambs. Values are means ± SEM. Significant differences ( P ≤ 0.05): * vs. CN.
    Figure Legend Snippet: Immunolocalization and protein expression of VEGF and HIF in lung tissue. Representative micrographs (40x) showing immunohistochemical distribution of VEGF-A in vascular wall of lung resistance arteries (A) , and analysis for VEGF-A vascular immunoreactivity intensity (B) . Scale bar: 100 μm. Scanned photograph of immunoblots (C) , and HIF-1α (D) and VEGF (E) protein expression. Protein expression was referred to β-actin as control protein. Groups are control (CN, open bars, n = 5) and melatonin treated (MN, closed bars, n = 5) lambs. Values are means ± SEM. Significant differences ( P ≤ 0.05): * vs. CN.

    Techniques Used: Expressing, Immunohistochemistry, Western Blot

    35) Product Images from "Astrocyte-derived VEGF-A drives blood-brain barrier disruption in CNS inflammatory disease"

    Article Title: Astrocyte-derived VEGF-A drives blood-brain barrier disruption in CNS inflammatory disease

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI60842

    Effects of VEGF-A on CLN-5 are mediated via eNOS.
    Figure Legend Snippet: Effects of VEGF-A on CLN-5 are mediated via eNOS.

    Techniques Used:

    GfapCre:Hif1a fl/fl mice show normal VEGF-A expression and BBB opening in inflammatory lesions.
    Figure Legend Snippet: GfapCre:Hif1a fl/fl mice show normal VEGF-A expression and BBB opening in inflammatory lesions.

    Techniques Used: Mouse Assay, Expressing

    Efficient inactivation of VEGF-A in the inflamed CNS in GfapCre:Vegfa fl/fl mice.
    Figure Legend Snippet: Efficient inactivation of VEGF-A in the inflamed CNS in GfapCre:Vegfa fl/fl mice.

    Techniques Used: Mouse Assay

    36) Product Images from "Gut microbiota regulates lacteal integrity by inducing VEGF‐C in intestinal villus macrophages"

    Article Title: Gut microbiota regulates lacteal integrity by inducing VEGF‐C in intestinal villus macrophages

    Journal: EMBO Reports

    doi: 10.15252/embr.201846927

    VEGF
    Figure Legend Snippet: VEGF

    Techniques Used:

    VEGF
    Figure Legend Snippet: VEGF

    Techniques Used:

    VEGF
    Figure Legend Snippet: VEGF

    Techniques Used:

    37) Product Images from "A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in angiogenesis"

    Article Title: A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in angiogenesis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03278-w

    VEGFR regulates angiogenesis and tumor VM through YAP/TAZ in vitro. a Transient knockdown of YAP and/or TAZ in MCF10A overexpressing VEGFR2 decreases expression of ANG-2 and CYR61. Western blotting exposures indicate relative expression of YAP and TAZ. b , c VEGFR2 overexpression and VEGF treatment increases tube formation by MCF10A through YAP/TAZ. YAP and/or TAZ were transiently knocked down by siRNA in MCF10A stably overexpressing VEGFR2 and subjected to tube-formation assay on Matrigel 48 h after transfection alongside wild type MCF10A. For some conditions, cells were stimulated with 100 ng ml −1 VEGF or were treated with 100 nM verteporfin for the duration of the tube formation assay. Representative images are shown in b . Scale bar denotes 200 μm. Total tube formation was quantified in c ( n = 3). * p
    Figure Legend Snippet: VEGFR regulates angiogenesis and tumor VM through YAP/TAZ in vitro. a Transient knockdown of YAP and/or TAZ in MCF10A overexpressing VEGFR2 decreases expression of ANG-2 and CYR61. Western blotting exposures indicate relative expression of YAP and TAZ. b , c VEGFR2 overexpression and VEGF treatment increases tube formation by MCF10A through YAP/TAZ. YAP and/or TAZ were transiently knocked down by siRNA in MCF10A stably overexpressing VEGFR2 and subjected to tube-formation assay on Matrigel 48 h after transfection alongside wild type MCF10A. For some conditions, cells were stimulated with 100 ng ml −1 VEGF or were treated with 100 nM verteporfin for the duration of the tube formation assay. Representative images are shown in b . Scale bar denotes 200 μm. Total tube formation was quantified in c ( n = 3). * p

    Techniques Used: In Vitro, Expressing, Western Blot, Over Expression, Stable Transfection, Tube Formation Assay, Transfection

    YAP/TAZ are mediators of VEGF-induced angiogenesis ex vivo and in vivo. a – c Pharmacological inhibition of YAP/TAZ reduces angiogenesis ex vivo in a rat aorta model. Sections of aorta were cultured for 7 days in Matrigel with 100 ng ml −1 VEGF and the indicated concentrations of VP. Representative images are shown in a . Sprout area is quantified in b . Scale bar denotes 500 μm. c Immunostaining of aorta sections demonstrates that outgrowths are positive for VE-cadherin. Scale bar denotes 300 μm. Wimasis image analysis software was used to visualize sprouts ( n = 3). d Transient knockdown of YAP/TAZ or pharmacological inhibition of YAP/TAZ with VP reduces angiogenesis by HUVECs in vivo in Matrigel plug experiments. Five million cells were injected subcutaneously into mice with Matrigel and 200 ng mL −1 VEGF. VP was administered by intraperitoneal injection every other day. Plugs were excised after 1 week. Representative images are shown in the top two rows. In the bottom panels, angiogenesis in the Matrigel plugs was stained by IHC for the human endothelial cell marker hCD31. Scale bar denotes 500 μm. e YAP/TAZ inhibition reduces endogenous angiogenesis in vivo in Matrigel plug experiments. Matrigel plugs with 200 ng ml −1 VEGF were implanted subcutaneously in mice. VP was administered by intraperitoneal injection every other day. Plugs were excised after 2 weeks. Angiogenesis was assessed by IHC staining for mouse mCD31 endothelial cell marker. Scale bar denotes 500 μm. f – h YAP/TAZ inhibition diminishes angiogenesis in vivo in a mouse retinal model. Mice were injected with 1 mg kg −1 VEGF with or without 100 mg kg −1 VP at postnatal day 3 (P3) and 4 (P4). At P5, retinal blood vasculature was stained. Representative images of the retinal vessel density are shown in f , whereas g shows images of the vascular front. Scale bar denotes 100 μm f or 30 μm g . Number of filopodia (active angiogenesis) is quantified in h . Each data point represents the average of two retinas from a single mouse ( n = 4 for control, n = 3 for VP). * p
    Figure Legend Snippet: YAP/TAZ are mediators of VEGF-induced angiogenesis ex vivo and in vivo. a – c Pharmacological inhibition of YAP/TAZ reduces angiogenesis ex vivo in a rat aorta model. Sections of aorta were cultured for 7 days in Matrigel with 100 ng ml −1 VEGF and the indicated concentrations of VP. Representative images are shown in a . Sprout area is quantified in b . Scale bar denotes 500 μm. c Immunostaining of aorta sections demonstrates that outgrowths are positive for VE-cadherin. Scale bar denotes 300 μm. Wimasis image analysis software was used to visualize sprouts ( n = 3). d Transient knockdown of YAP/TAZ or pharmacological inhibition of YAP/TAZ with VP reduces angiogenesis by HUVECs in vivo in Matrigel plug experiments. Five million cells were injected subcutaneously into mice with Matrigel and 200 ng mL −1 VEGF. VP was administered by intraperitoneal injection every other day. Plugs were excised after 1 week. Representative images are shown in the top two rows. In the bottom panels, angiogenesis in the Matrigel plugs was stained by IHC for the human endothelial cell marker hCD31. Scale bar denotes 500 μm. e YAP/TAZ inhibition reduces endogenous angiogenesis in vivo in Matrigel plug experiments. Matrigel plugs with 200 ng ml −1 VEGF were implanted subcutaneously in mice. VP was administered by intraperitoneal injection every other day. Plugs were excised after 2 weeks. Angiogenesis was assessed by IHC staining for mouse mCD31 endothelial cell marker. Scale bar denotes 500 μm. f – h YAP/TAZ inhibition diminishes angiogenesis in vivo in a mouse retinal model. Mice were injected with 1 mg kg −1 VEGF with or without 100 mg kg −1 VP at postnatal day 3 (P3) and 4 (P4). At P5, retinal blood vasculature was stained. Representative images of the retinal vessel density are shown in f , whereas g shows images of the vascular front. Scale bar denotes 100 μm f or 30 μm g . Number of filopodia (active angiogenesis) is quantified in h . Each data point represents the average of two retinas from a single mouse ( n = 4 for control, n = 3 for VP). * p

    Techniques Used: Ex Vivo, In Vivo, Inhibition, Cell Culture, Immunostaining, Software, Injection, Mouse Assay, Staining, Immunohistochemistry, Marker

    Model for VEGFR and Hippo signaling in angiogenesis/VM. When VEGF binds to its receptor, VEGFR, signaling through PI3K and MAPK is initiated. This leads to the inhibition of MST/LATS and subsequent activation of YAP/TAZ. YAP and TAZ induce ANG-2 and CYR61 expression, leading to enhanced angiogenesis and vasculogenic mimicry in endothelial and tumor cell lines, respectively
    Figure Legend Snippet: Model for VEGFR and Hippo signaling in angiogenesis/VM. When VEGF binds to its receptor, VEGFR, signaling through PI3K and MAPK is initiated. This leads to the inhibition of MST/LATS and subsequent activation of YAP/TAZ. YAP and TAZ induce ANG-2 and CYR61 expression, leading to enhanced angiogenesis and vasculogenic mimicry in endothelial and tumor cell lines, respectively

    Techniques Used: Inhibition, Microscale Thermophoresis, Activation Assay, Expressing

    VEGFR is an upstream regulator of Hippo signaling. VEGFR inhibition activates LATS-BS a and suppresses YAP/TAZ transcriptional co-activation in HEK293A b . Cells were treated with each inhibitor for 4 h at 10 μM ( n = 3). c VEGFR inhibition diminishes expression of YAP/TAZ targets, CYR61/CTGF . HEK293A were treated with inhibitors for 4 h at 10 μM. CYR61 or CTGF mRNA expression was determined by qRT-PCR ( n = 3). d VEGFR reduces YAP-S127 phosphorylation in HEK293 (western blotting). e VEGF stimulation inhibits LATS-BS activity in MCF10A stably overexpressing VEGFR1/2. MCF10A-VEGFR1/2 were transfected with LATS-BS. Cells were treated with VEGF (100 ng ml −1 ) for the indicated times. For some samples, cells were pre-treated with axitinib at 10 μM for 3 h before VEGF treatment ( n = 3). f , g VEGF increases YAP/TAZ transcriptional co-activation of CYR61 in MCF10A-VEGFR1/2 ( f ), as well as in BOEC and MDA-MB231 ( g ). Cells were treated with 100 ng ml −1 VEGF for the indicated times. CYR61 expression was measured by qRT-PCR. For some samples, cells were pre-treated with Axitinib at 10 μM for 3 h before VEGF treatment ( n = 3). h - m VEGF stimulation increases YAP/TAZ nuclear localization in MCF10A-VEGFR2 ( h , i ), MDA-MB231 ( j , k ), and BOEC ( l , m ). h , j , l Representative images of YAP or TAZ immunostaining are shown after treatment with 100 ng ml −1 VEGF for the indicated times. Scale bar represents 15 μm. i , k , m YAP/TAZ subcellular localization was quantified in three separate experiments in which at least 200 cells were examined. n , o VEGFR2 signals through PI3K, AKT, and MEK to inhibit LATS-BS ( n ) and activate the STBS reporter ( o ). Cells were untreated or treated with VEGFR inhibitor (axitinib), PI3K inhibitor (LY294002), AKT inhibitor (triciribine), or MEK inhibitor (PD98059) at 10 μM for 4 h ( n = 3). * p
    Figure Legend Snippet: VEGFR is an upstream regulator of Hippo signaling. VEGFR inhibition activates LATS-BS a and suppresses YAP/TAZ transcriptional co-activation in HEK293A b . Cells were treated with each inhibitor for 4 h at 10 μM ( n = 3). c VEGFR inhibition diminishes expression of YAP/TAZ targets, CYR61/CTGF . HEK293A were treated with inhibitors for 4 h at 10 μM. CYR61 or CTGF mRNA expression was determined by qRT-PCR ( n = 3). d VEGFR reduces YAP-S127 phosphorylation in HEK293 (western blotting). e VEGF stimulation inhibits LATS-BS activity in MCF10A stably overexpressing VEGFR1/2. MCF10A-VEGFR1/2 were transfected with LATS-BS. Cells were treated with VEGF (100 ng ml −1 ) for the indicated times. For some samples, cells were pre-treated with axitinib at 10 μM for 3 h before VEGF treatment ( n = 3). f , g VEGF increases YAP/TAZ transcriptional co-activation of CYR61 in MCF10A-VEGFR1/2 ( f ), as well as in BOEC and MDA-MB231 ( g ). Cells were treated with 100 ng ml −1 VEGF for the indicated times. CYR61 expression was measured by qRT-PCR. For some samples, cells were pre-treated with Axitinib at 10 μM for 3 h before VEGF treatment ( n = 3). h - m VEGF stimulation increases YAP/TAZ nuclear localization in MCF10A-VEGFR2 ( h , i ), MDA-MB231 ( j , k ), and BOEC ( l , m ). h , j , l Representative images of YAP or TAZ immunostaining are shown after treatment with 100 ng ml −1 VEGF for the indicated times. Scale bar represents 15 μm. i , k , m YAP/TAZ subcellular localization was quantified in three separate experiments in which at least 200 cells were examined. n , o VEGFR2 signals through PI3K, AKT, and MEK to inhibit LATS-BS ( n ) and activate the STBS reporter ( o ). Cells were untreated or treated with VEGFR inhibitor (axitinib), PI3K inhibitor (LY294002), AKT inhibitor (triciribine), or MEK inhibitor (PD98059) at 10 μM for 4 h ( n = 3). * p

    Techniques Used: Inhibition, Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, Stable Transfection, Transfection, Multiple Displacement Amplification, Immunostaining

    38) Product Images from "miR29b regulates aberrant methylation in In-Vitro diabetic nephropathy model of renal proximal tubular cells"

    Article Title: miR29b regulates aberrant methylation in In-Vitro diabetic nephropathy model of renal proximal tubular cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208044

    Effect of high glucose, angiotensin II and AGE on RPTECs. A and B: Assessment of mRNA expression profiles and protein levels of TGF-β1 in control, HG and cells treated with HG (30 mM), Ang II (1 μM) and AGE products (150 μg/ml) respectively. C and D : Assessment of mRNA expression profiles and protein levels of VEGF-A in control and cells treated with HG (30 mM), Ang II (1 μM) and AGE product (150 μg/ml) respectively and E: Assessment of mRNA level and protein level of Col4A1 in control and cells treated with HG (30 mM), Ang II (1 μM) and AGE product (150 μg/ml) respectively. Results are represented as mean ± S.D. (n = 3) , *p
    Figure Legend Snippet: Effect of high glucose, angiotensin II and AGE on RPTECs. A and B: Assessment of mRNA expression profiles and protein levels of TGF-β1 in control, HG and cells treated with HG (30 mM), Ang II (1 μM) and AGE products (150 μg/ml) respectively. C and D : Assessment of mRNA expression profiles and protein levels of VEGF-A in control and cells treated with HG (30 mM), Ang II (1 μM) and AGE product (150 μg/ml) respectively and E: Assessment of mRNA level and protein level of Col4A1 in control and cells treated with HG (30 mM), Ang II (1 μM) and AGE product (150 μg/ml) respectively. Results are represented as mean ± S.D. (n = 3) , *p

    Techniques Used: Expressing

    39) Product Images from "Structural Basis for Selective Vascular Endothelial Growth Factor-A (VEGF-A) Binding to Neuropilin-1"

    Article Title: Structural Basis for Selective Vascular Endothelial Growth Factor-A (VEGF-A) Binding to Neuropilin-1

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.331140

    Crystal structure of VEGF-A HBD in complex with Nrp1. A , chain A ( green ) and chain B ( blue ) crystallized in an antiparallel fashion with the chain A VEGF-A HBD fully engaging the Nrp1-b1 domain of chain B, and that of chain B engaged by the symmetry related
    Figure Legend Snippet: Crystal structure of VEGF-A HBD in complex with Nrp1. A , chain A ( green ) and chain B ( blue ) crystallized in an antiparallel fashion with the chain A VEGF-A HBD fully engaging the Nrp1-b1 domain of chain B, and that of chain B engaged by the symmetry related

    Techniques Used:

    Glu-154 of VEGF-A 164 exon 7 contributes to Nrp1 binding. A , Glu-154 interacts with the side chain hydroxyl (bond distance = 2.73 Å) and backbone amide (bond distance = 3.16 Å) of Thr-299 of the Nrp1 L1 loop. B , mutation of Glu-154 to alanine
    Figure Legend Snippet: Glu-154 of VEGF-A 164 exon 7 contributes to Nrp1 binding. A , Glu-154 interacts with the side chain hydroxyl (bond distance = 2.73 Å) and backbone amide (bond distance = 3.16 Å) of Thr-299 of the Nrp1 L1 loop. B , mutation of Glu-154 to alanine

    Techniques Used: Binding Assay, Mutagenesis

    VEGF-A 164 binds to Nrp1 with high affinity. VEGF-A 164 ( black line ) binds Nrp1 with a K d = 3.0 n m ± 0.2 n m . VEGF-A 120 ( gray dashed line ) binds Nrp1 with a K d = 22 n m ± 1 n m .
    Figure Legend Snippet: VEGF-A 164 binds to Nrp1 with high affinity. VEGF-A 164 ( black line ) binds Nrp1 with a K d = 3.0 n m ± 0.2 n m . VEGF-A 120 ( gray dashed line ) binds Nrp1 with a K d = 22 n m ± 1 n m .

    Techniques Used:

    Exon 7-encoded residues of VEGF-A 164 are responsible for Nrp1 binding selectivity. A , superimposition of Nrp1 (PDB code 1KEX ) and Nrp2 b1 domains (PDB code 2QQJ , residues 276–427) reveals a similar overall architecture, root mean square deviation
    Figure Legend Snippet: Exon 7-encoded residues of VEGF-A 164 are responsible for Nrp1 binding selectivity. A , superimposition of Nrp1 (PDB code 1KEX ) and Nrp2 b1 domains (PDB code 2QQJ , residues 276–427) reveals a similar overall architecture, root mean square deviation

    Techniques Used: Binding Assay

    Exon 8-encoded C-terminal arginine of VEGF-A mediates high affinity Nrp1 binding. A , Arg-164 forms specific contacts with the b1 binding pocket of Nrp1. The guanidinium moiety forms a salt bridge with Asp-320 carboxylate oxygens ( dashed red lines , 3.08
    Figure Legend Snippet: Exon 8-encoded C-terminal arginine of VEGF-A mediates high affinity Nrp1 binding. A , Arg-164 forms specific contacts with the b1 binding pocket of Nrp1. The guanidinium moiety forms a salt bridge with Asp-320 carboxylate oxygens ( dashed red lines , 3.08

    Techniques Used: Binding Assay

    HBD of VEGF-A is responsible for selective binding to the Nrp1 b1 domain. Exon 8-encoded residues mediate high affinity binding whereas exon 7-encoded residues primarily govern selectivity.
    Figure Legend Snippet: HBD of VEGF-A is responsible for selective binding to the Nrp1 b1 domain. Exon 8-encoded residues mediate high affinity binding whereas exon 7-encoded residues primarily govern selectivity.

    Techniques Used: Binding Assay

    40) Product Images from "Ca2+ Influx through Reverse Mode Na+/Ca2+ Exchange Is Critical for Vascular Endothelial Growth Factor-mediated Extracellular Signal-regulated Kinase (ERK) 1/2 Activation and Angiogenic Functions of Human Endothelial Cells *"

    Article Title: Ca2+ Influx through Reverse Mode Na+/Ca2+ Exchange Is Critical for Vascular Endothelial Growth Factor-mediated Extracellular Signal-regulated Kinase (ERK) 1/2 Activation and Angiogenic Functions of Human Endothelial Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.251777

    Effect of reverse mode NCX inhibitors on Ca 2+ transients. Representative time courses of Ca 2+ -sensitive Fluo-4NW fluorescence recorded during VEGF stimulation of HUVECs are shown. Serum-starved HUVECs were stimulated with VEGF (50 ng/ml) at time 0. A–C
    Figure Legend Snippet: Effect of reverse mode NCX inhibitors on Ca 2+ transients. Representative time courses of Ca 2+ -sensitive Fluo-4NW fluorescence recorded during VEGF stimulation of HUVECs are shown. Serum-starved HUVECs were stimulated with VEGF (50 ng/ml) at time 0. A–C

    Techniques Used: Fluorescence

    Effect of NCX siRNA on phospho-ERK1/2 and HUVEC proliferation. A , HUVECs transfected with control nontargeting siRNA or NCX1 targeting siRNA (100 n m for 48 h) were serum-starved and subsequently challenged with VEGF (50 ng/ml) for 10 min. ERK1/2 and PLCγ
    Figure Legend Snippet: Effect of NCX siRNA on phospho-ERK1/2 and HUVEC proliferation. A , HUVECs transfected with control nontargeting siRNA or NCX1 targeting siRNA (100 n m for 48 h) were serum-starved and subsequently challenged with VEGF (50 ng/ml) for 10 min. ERK1/2 and PLCγ

    Techniques Used: Transfection

    Proposed mechanism of reverse mode NCX involvement in VEGF-induced ERK1/2 phosphorylation. Bold lines show direct interactions. Dashed lines show indirect interactions. Activation of PLCγ downstream of VEGFR leads to the generation of diacylglycerol
    Figure Legend Snippet: Proposed mechanism of reverse mode NCX involvement in VEGF-induced ERK1/2 phosphorylation. Bold lines show direct interactions. Dashed lines show indirect interactions. Activation of PLCγ downstream of VEGFR leads to the generation of diacylglycerol

    Techniques Used: Activation Assay

    Effect of NCX inhibitors on angiogenesis functional assays. A , HUVECs were exposed to VEGF (50 ng/ml) for 48 h in the presence of the indicated concentrations of DCB and KBR-7943, in MCDB-131 containing 0.1%w/v BSA. Bar C , control; bar Am , amiloride.
    Figure Legend Snippet: Effect of NCX inhibitors on angiogenesis functional assays. A , HUVECs were exposed to VEGF (50 ng/ml) for 48 h in the presence of the indicated concentrations of DCB and KBR-7943, in MCDB-131 containing 0.1%w/v BSA. Bar C , control; bar Am , amiloride.

    Techniques Used: Functional Assay

    Effect of NCX inhibitors on VEGF-induced ERK1/2 phosphorylation. A , serum-starved HUVECs were preincubated (for 30 min) with 30 μ m DCB or vehicle (0.5% v/v Me 2 SO) prior to VEGF stimulation (50 ng/ml) for the times indicated. B , ERK1/2 and PLCγ
    Figure Legend Snippet: Effect of NCX inhibitors on VEGF-induced ERK1/2 phosphorylation. A , serum-starved HUVECs were preincubated (for 30 min) with 30 μ m DCB or vehicle (0.5% v/v Me 2 SO) prior to VEGF stimulation (50 ng/ml) for the times indicated. B , ERK1/2 and PLCγ

    Techniques Used:

    Related Articles

    Enzyme-linked Immunosorbent Assay:

    Article Title: Microenvironment Changes (in pH) Affect VEGF Alternative Splicing
    Article Snippet: .. ELISA, Protein Extraction and Western Blotting Culture supernatants from RL95 in different conditions were collected and used to measure human VEGF by ELISA (Oncogene Research Products) under conditions described by the supplier. ..

    Protein Extraction:

    Article Title: Microenvironment Changes (in pH) Affect VEGF Alternative Splicing
    Article Snippet: .. ELISA, Protein Extraction and Western Blotting Culture supernatants from RL95 in different conditions were collected and used to measure human VEGF by ELISA (Oncogene Research Products) under conditions described by the supplier. ..

    other:

    Article Title: Lysosomal Pathways and Autophagy Distinctively Control Endothelial Cell Behavior to Affect Tumor Vasculature
    Article Snippet: Methyl cellulose (M6385), VEGF-A (SRP-3182), N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (D5642), paraformaldehyde (P6148), DMSO (472301), Leupeptin (L-2884) and chloroquine diphosphate salt (C6628) were from Sigma-Aldrich (Bornem, Belgium).

    Expressing:

    Article Title: AAVrh.10-mediated Genetic Delivery of Bevacizumab to the Pleura to Provide Local Anti-VEGF to Suppress Growth of Metastatic Lung Tumors
    Article Snippet: .. Supernatants were concentrated by passage through Ultracel YM-10 centrifugal filters (Millipore, Billerica, MA) and evaluated for the expression of anti-human VEGF-A antibody by Western analysis under reducing conditions, using a peroxidase-conjugated sheep anti-mouse IgG secondary antibody (SIGMA, Saint Louis, MO) and enhanced chemiluminescence (ECL) reagent (GE Healthcare Life Sciences, Piscataway, NJ). .. The specificity of the AAVrh.10-expressed anti-VEGF antibody for human VEGF was determined by Western analysis with human VEGF-A121 and VEGF-A165 or with mouse VEGF-A120 and VEGF-A164 as the target antigens and infected cell supernatants as the primary antibody.

    Western Blot:

    Article Title: AAVrh.10-mediated Genetic Delivery of Bevacizumab to the Pleura to Provide Local Anti-VEGF to Suppress Growth of Metastatic Lung Tumors
    Article Snippet: .. Supernatants were concentrated by passage through Ultracel YM-10 centrifugal filters (Millipore, Billerica, MA) and evaluated for the expression of anti-human VEGF-A antibody by Western analysis under reducing conditions, using a peroxidase-conjugated sheep anti-mouse IgG secondary antibody (SIGMA, Saint Louis, MO) and enhanced chemiluminescence (ECL) reagent (GE Healthcare Life Sciences, Piscataway, NJ). .. The specificity of the AAVrh.10-expressed anti-VEGF antibody for human VEGF was determined by Western analysis with human VEGF-A121 and VEGF-A165 or with mouse VEGF-A120 and VEGF-A164 as the target antigens and infected cell supernatants as the primary antibody.

    Article Title: Differential Healing After Sirolimus, Paclitaxel and Bare Metal Stent Placement in Combination with PPARγ Agonists: Requirement for mTOR/Akt2 in PPARγ Activation
    Article Snippet: .. Paclitaxel and human VEGF were purchased from Sigma Antibodies used for Western blot were anti-phospho-Ser 2448 mTOR, anti-mTOR, anti-phospho-Thr 421/Ser424 p70 S6k Kinase, anti-p70 S6 Kinase, anti-phospho-Ser-65 4E-BP-1, anti-4E-BP-1, anti-phospho-Ser-473-Akt, anti-Akt all purchased from Cell Signaling (Beverly, MA). .. Anti β-actin was purchased from Abcam (Cambridge, MA).

    Article Title: Microenvironment Changes (in pH) Affect VEGF Alternative Splicing
    Article Snippet: .. ELISA, Protein Extraction and Western Blotting Culture supernatants from RL95 in different conditions were collected and used to measure human VEGF by ELISA (Oncogene Research Products) under conditions described by the supplier. ..

    Recombinant:

    Article Title: Protein Phosphotyrosine Phosphatase 1B (PTP1B) in Calpain-dependent Feedback Regulation of Vascular Endothelial Growth Factor Receptor (VEGFR2) in Endothelial Cells
    Article Snippet: .. Treatments were applied directly on the wound bed in PBS at total volume of 5 μl with or without recombinant human VEGF (200 ng) , ALLN (20 nmol), and PTP1B inhibitor (50 nmol, Calbiochem). .. Five micrograms of HA-tagged PTP1B and human calpain 1 and calpain 2 (2.5 μg each) plasmids were prepared with a lipid-based in vivo transfection kit and intracutaneously injected near the wound bed.

    Inhibition:

    Article Title: Adipokines and Vascular Endothelial Growth Factor in Normal Human Breast Tissue in Vivo – Correlations and Attenuation by Dietary Flaxseed
    Article Snippet: .. For the inhibition assays co-cultures were treated with anti-human leptin antibody (Genway, USA), anti-human VEGF antibody (Calbiochem, USA), or normal IgG from the same species (R & D system, USA) at 0.1 μg/ml and 1 μg/ml in presence or absence of 10−9 M estradiol. ..

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  • 88
    Millipore antibodies against vegf c
    Expression of <t>VEGF-C</t> and miR-27b, miR-101 or miR-128 and their correlation with patients’ survival in gastric cancers Kaplan-Meier survival analysis and Log-rank test showed that the patients with higher VEGF-C expression had a poorer overall survival and disease-free survival. A. and B. However, there was no significant difference in the overall survival and disease-free survival between the miR-27b C. and D. , miR-101 E. and F. or miR-128 G. and H. lower and higher expression groups.
    Antibodies Against Vegf C, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore vegf a
    Effects of ABL on <t>VEGF-induced</t> HUVEC migration and tube formation. A-B) ABL increases VEGF-induced ECs transwell migration. The data represent the number of migrated cells per microscopic field in VEGF-treated cells vs. control cells. C-D) ABL increases VEGF-induced EC transwell migration. Confluent HUVECs in a monolayer were pretreated with ABL or vehicle for 2 h and wounded with a cell scraper. After 48 h of incubation at 37°C, the number of cells that migrated across the wound edge was counted in each field. E-F) ABL increases VEGF-induced EC tube formation. The images were visualized using a phase contrast microscope (10× magnification). The total tubule length from 10 non-overlapping fields was measured by tracing the tube-like structure. The values represent the mean ± SEM from 3 independent experiments ( n = 3). Scale bar: 100 μm.
    Vegf A, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore vegf mrna
    <t>VEGF</t> siRNA-generating pDNA down regulates VEGF <t>mRNA</t> levels in rat cervix . The VEGF gene silencing efficiency of the selected siRNA-generating pDNA was tested in vivo in the cervix of pregnant rat from GD17–20. VEGF siRNA-generating pDNA was found to down-regulate VEGF compared to control. n = 5, ( p
    Vegf Mrna, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Expression of VEGF-C and miR-27b, miR-101 or miR-128 and their correlation with patients’ survival in gastric cancers Kaplan-Meier survival analysis and Log-rank test showed that the patients with higher VEGF-C expression had a poorer overall survival and disease-free survival. A. and B. However, there was no significant difference in the overall survival and disease-free survival between the miR-27b C. and D. , miR-101 E. and F. or miR-128 G. and H. lower and higher expression groups.

    Journal: Oncotarget

    Article Title: MicroRNA-27b, microRNA-101 and microRNA-128 inhibit angiogenesis by down-regulating vascular endothelial growth factor C expression in gastric cancers

    doi:

    Figure Lengend Snippet: Expression of VEGF-C and miR-27b, miR-101 or miR-128 and their correlation with patients’ survival in gastric cancers Kaplan-Meier survival analysis and Log-rank test showed that the patients with higher VEGF-C expression had a poorer overall survival and disease-free survival. A. and B. However, there was no significant difference in the overall survival and disease-free survival between the miR-27b C. and D. , miR-101 E. and F. or miR-128 G. and H. lower and higher expression groups.

    Article Snippet: Western blot Forty-eight hours after transfection with the miRNA mimics or the negative control, total protein was extracted from the transfected cells, incubated with primary antibodies against VEGF-C (1:300, Sangon) or β-actin (1:500, Zhongshan) overnight at 4°C, washed, and then detected with a chemiluminescence kit (Millipore, Billerica, MA, USA) according to the manufacturer's procedure.

    Techniques: Expressing

    Overexpression of miR-27b, miR-101, or miR-128 attenuated proliferation and tube formation of HUVECs The secretion level of VEGF-C significantly decreased in the culture supernatant from the miRNA-transfected cells, compared to the negative control groups, determined by ELISA (A1). The percentages of proliferating cells were quantified by EdU incorporation experiments. Decreased proliferation activity of the miRNAs-transfected groups was observed compared to the negative control groups. (A2). Significantly inverse correlation was found between VEGF-C expression and MVD (A3) or LVD (A4) in human gastric cancers samples. The number of HUVECs decreased by treatment with the medium supernatant from the miRNAs-transfected groups compared to the negative groups B. . Proliferating HUVECs were labeled after conjugated reaction of Apollo dye and EdU (red). Cell nuclei stained with DAPI (blue) represents a total population of cells. The images are representative of the results obtained C. , with the quantifiable results shown in Figure 5A2 . Tube formation of endothelial cells was dramatically inhibited in the transfected groups compared to the negative groups D. . * P

    Journal: Oncotarget

    Article Title: MicroRNA-27b, microRNA-101 and microRNA-128 inhibit angiogenesis by down-regulating vascular endothelial growth factor C expression in gastric cancers

    doi:

    Figure Lengend Snippet: Overexpression of miR-27b, miR-101, or miR-128 attenuated proliferation and tube formation of HUVECs The secretion level of VEGF-C significantly decreased in the culture supernatant from the miRNA-transfected cells, compared to the negative control groups, determined by ELISA (A1). The percentages of proliferating cells were quantified by EdU incorporation experiments. Decreased proliferation activity of the miRNAs-transfected groups was observed compared to the negative control groups. (A2). Significantly inverse correlation was found between VEGF-C expression and MVD (A3) or LVD (A4) in human gastric cancers samples. The number of HUVECs decreased by treatment with the medium supernatant from the miRNAs-transfected groups compared to the negative groups B. . Proliferating HUVECs were labeled after conjugated reaction of Apollo dye and EdU (red). Cell nuclei stained with DAPI (blue) represents a total population of cells. The images are representative of the results obtained C. , with the quantifiable results shown in Figure 5A2 . Tube formation of endothelial cells was dramatically inhibited in the transfected groups compared to the negative groups D. . * P

    Article Snippet: Western blot Forty-eight hours after transfection with the miRNA mimics or the negative control, total protein was extracted from the transfected cells, incubated with primary antibodies against VEGF-C (1:300, Sangon) or β-actin (1:500, Zhongshan) overnight at 4°C, washed, and then detected with a chemiluminescence kit (Millipore, Billerica, MA, USA) according to the manufacturer's procedure.

    Techniques: Over Expression, Transfection, Negative Control, Enzyme-linked Immunosorbent Assay, Activity Assay, Expressing, Labeling, Staining

    Immunohistochemical staining of VEGF-C, blood vessels, and lymphatic vessels in gastric cancers VEGF-C immunoreactivity was classified as three grades: weak A. , moderate B. and strong C. (× 400). The blood vessels were clearly distinguished from lymphatic vessels by double immunohistochemical staining for CD-34 (red, blank arrowhead) and D2-40 (brown, red arrow). The tumor emboli in the lymphatic vessel was also observed (yellow triangular arrowhead) D. , (× 400).

    Journal: Oncotarget

    Article Title: MicroRNA-27b, microRNA-101 and microRNA-128 inhibit angiogenesis by down-regulating vascular endothelial growth factor C expression in gastric cancers

    doi:

    Figure Lengend Snippet: Immunohistochemical staining of VEGF-C, blood vessels, and lymphatic vessels in gastric cancers VEGF-C immunoreactivity was classified as three grades: weak A. , moderate B. and strong C. (× 400). The blood vessels were clearly distinguished from lymphatic vessels by double immunohistochemical staining for CD-34 (red, blank arrowhead) and D2-40 (brown, red arrow). The tumor emboli in the lymphatic vessel was also observed (yellow triangular arrowhead) D. , (× 400).

    Article Snippet: Western blot Forty-eight hours after transfection with the miRNA mimics or the negative control, total protein was extracted from the transfected cells, incubated with primary antibodies against VEGF-C (1:300, Sangon) or β-actin (1:500, Zhongshan) overnight at 4°C, washed, and then detected with a chemiluminescence kit (Millipore, Billerica, MA, USA) according to the manufacturer's procedure.

    Techniques: Immunohistochemistry, Staining

    MiR-27b, miR-101, or miR-128 directly down-regulates VEGF-C expression through posttranscriptional repression in gastric cancer cells Scheme representation of the potential binding site of miR-27b, miR-101, or miR-128 in the VEGF-C 3′UTR A. . Dual-luciferase reporter gene assay showed that miR-27b, miR-101, or miR-128(decreased 38.68% ± 10.86%, 30.36% ± 10.29%, 47.76% ± 13.61%, p = 0.0115, p = 0.0156, or p = 0.0111) respectively, but not miR-144 or miR-186 displayed strong inhibitory effect on the luciferases expression in MKN-45 cells. B. . MiR-27b, miR-101, miR-128, miR-27b/miR-101, miR-27b/miR-128 or miR-101/miR-128 co-transfection could significantly suppress the luciferase activity in pmiR-VEGF-C transfected MKN-45 cells C. . MiR-27b, miR-101, miR-128 or miR-27b/miR-101, miR-27b/miR-128, miR-101/miR-128 co-transfection could significantly reduce the VEGF-C mRNA expression in MKN-45 cells D. . MiR-27b, miR-101, miR-128 or miR-27b/miR-101, miR-27b/miR-128, miR-101/miR-128 co-transfection could significantly decrease the VEGF-C protein expression in MKN-45 cells E. . Compared to human non-tumorous gastric mucosa ( n = 5), higher expression of VEGF-C mRNA and protein and decreased expression of miR-27b, miR-101 or miR-128 were detected in 3 gastric cancer cell lines by Western blot and RT-qPCR, respectively F. . Decreased miR-27b, miR-101, or miR-128 levels were found in gastric cancer tissues compared to the non-tumorous gastric mucosae (G1). An inverse correlation was found between miR-27b (G2) and miR-128 (G4) expression and VEGF-C levels in human gastric cancers samples. However, there was no significantly correlation between miR-101 level and VEGF-C expression (G3). * P

    Journal: Oncotarget

    Article Title: MicroRNA-27b, microRNA-101 and microRNA-128 inhibit angiogenesis by down-regulating vascular endothelial growth factor C expression in gastric cancers

    doi:

    Figure Lengend Snippet: MiR-27b, miR-101, or miR-128 directly down-regulates VEGF-C expression through posttranscriptional repression in gastric cancer cells Scheme representation of the potential binding site of miR-27b, miR-101, or miR-128 in the VEGF-C 3′UTR A. . Dual-luciferase reporter gene assay showed that miR-27b, miR-101, or miR-128(decreased 38.68% ± 10.86%, 30.36% ± 10.29%, 47.76% ± 13.61%, p = 0.0115, p = 0.0156, or p = 0.0111) respectively, but not miR-144 or miR-186 displayed strong inhibitory effect on the luciferases expression in MKN-45 cells. B. . MiR-27b, miR-101, miR-128, miR-27b/miR-101, miR-27b/miR-128 or miR-101/miR-128 co-transfection could significantly suppress the luciferase activity in pmiR-VEGF-C transfected MKN-45 cells C. . MiR-27b, miR-101, miR-128 or miR-27b/miR-101, miR-27b/miR-128, miR-101/miR-128 co-transfection could significantly reduce the VEGF-C mRNA expression in MKN-45 cells D. . MiR-27b, miR-101, miR-128 or miR-27b/miR-101, miR-27b/miR-128, miR-101/miR-128 co-transfection could significantly decrease the VEGF-C protein expression in MKN-45 cells E. . Compared to human non-tumorous gastric mucosa ( n = 5), higher expression of VEGF-C mRNA and protein and decreased expression of miR-27b, miR-101 or miR-128 were detected in 3 gastric cancer cell lines by Western blot and RT-qPCR, respectively F. . Decreased miR-27b, miR-101, or miR-128 levels were found in gastric cancer tissues compared to the non-tumorous gastric mucosae (G1). An inverse correlation was found between miR-27b (G2) and miR-128 (G4) expression and VEGF-C levels in human gastric cancers samples. However, there was no significantly correlation between miR-101 level and VEGF-C expression (G3). * P

    Article Snippet: Western blot Forty-eight hours after transfection with the miRNA mimics or the negative control, total protein was extracted from the transfected cells, incubated with primary antibodies against VEGF-C (1:300, Sangon) or β-actin (1:500, Zhongshan) overnight at 4°C, washed, and then detected with a chemiluminescence kit (Millipore, Billerica, MA, USA) according to the manufacturer's procedure.

    Techniques: Expressing, Binding Assay, Luciferase, Reporter Gene Assay, Cotransfection, Activity Assay, Transfection, Western Blot, Quantitative RT-PCR

    Effects of ABL on VEGF-induced HUVEC migration and tube formation. A-B) ABL increases VEGF-induced ECs transwell migration. The data represent the number of migrated cells per microscopic field in VEGF-treated cells vs. control cells. C-D) ABL increases VEGF-induced EC transwell migration. Confluent HUVECs in a monolayer were pretreated with ABL or vehicle for 2 h and wounded with a cell scraper. After 48 h of incubation at 37°C, the number of cells that migrated across the wound edge was counted in each field. E-F) ABL increases VEGF-induced EC tube formation. The images were visualized using a phase contrast microscope (10× magnification). The total tubule length from 10 non-overlapping fields was measured by tracing the tube-like structure. The values represent the mean ± SEM from 3 independent experiments ( n = 3). Scale bar: 100 μm.

    Journal: PLoS ONE

    Article Title: Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells

    doi: 10.1371/journal.pone.0148968

    Figure Lengend Snippet: Effects of ABL on VEGF-induced HUVEC migration and tube formation. A-B) ABL increases VEGF-induced ECs transwell migration. The data represent the number of migrated cells per microscopic field in VEGF-treated cells vs. control cells. C-D) ABL increases VEGF-induced EC transwell migration. Confluent HUVECs in a monolayer were pretreated with ABL or vehicle for 2 h and wounded with a cell scraper. After 48 h of incubation at 37°C, the number of cells that migrated across the wound edge was counted in each field. E-F) ABL increases VEGF-induced EC tube formation. The images were visualized using a phase contrast microscope (10× magnification). The total tubule length from 10 non-overlapping fields was measured by tracing the tube-like structure. The values represent the mean ± SEM from 3 independent experiments ( n = 3). Scale bar: 100 μm.

    Article Snippet: The next day, pretreated the cells with vehicle or different doses of ABL, followed by exposure of some of the wells (n = 16) to VEGF-A (50 ng/mL) for 48 h. Cell growth was assessed using an MTT assay (Millipore Corporation, Temecula, CA, USA) according to the manufacturer’s protocol.

    Techniques: Migration, Incubation, Microscopy

    Schematic representation of the ABL effect on VEGF signaling in ECs. ABL inhibits VEGFR-2 and VE-cadherin association, thus promoting VEGFR-2 internalization, following by VEGF-induced VEGFR-2 phosphorylation and downstream signaling.

    Journal: PLoS ONE

    Article Title: Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells

    doi: 10.1371/journal.pone.0148968

    Figure Lengend Snippet: Schematic representation of the ABL effect on VEGF signaling in ECs. ABL inhibits VEGFR-2 and VE-cadherin association, thus promoting VEGFR-2 internalization, following by VEGF-induced VEGFR-2 phosphorylation and downstream signaling.

    Article Snippet: The next day, pretreated the cells with vehicle or different doses of ABL, followed by exposure of some of the wells (n = 16) to VEGF-A (50 ng/mL) for 48 h. Cell growth was assessed using an MTT assay (Millipore Corporation, Temecula, CA, USA) according to the manufacturer’s protocol.

    Techniques:

    ABL enhances VEGF signaling in endothelial cells. A) HUVECs were serum-starved overnight and pretreated with ABL or vehicle for 2 h, followed by exposure to VEGF-A (50 ng/mL) for various times as indicated. MAPK MAPK p44/42 Thr202/Tyr204 , p38 Thr180/Tyr182 , and Akt Ser473 phosphorylation in the cell lysates were measured via western blot analysis. B-D) The quantitative analysis represents the ratio of phosphorylated/total MAPK MAPK p44/42, MAPK p38, and Akt from 3 independent experiments. E) VEGFR-2 Tyr1175 phosphorylation in the cell lysates was measured via western blot analysis. F) Quantitative analysis of the ratio of phosphorylated/total VEGFR-2 from three independent experiments ( n = 3).

    Journal: PLoS ONE

    Article Title: Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells

    doi: 10.1371/journal.pone.0148968

    Figure Lengend Snippet: ABL enhances VEGF signaling in endothelial cells. A) HUVECs were serum-starved overnight and pretreated with ABL or vehicle for 2 h, followed by exposure to VEGF-A (50 ng/mL) for various times as indicated. MAPK MAPK p44/42 Thr202/Tyr204 , p38 Thr180/Tyr182 , and Akt Ser473 phosphorylation in the cell lysates were measured via western blot analysis. B-D) The quantitative analysis represents the ratio of phosphorylated/total MAPK MAPK p44/42, MAPK p38, and Akt from 3 independent experiments. E) VEGFR-2 Tyr1175 phosphorylation in the cell lysates was measured via western blot analysis. F) Quantitative analysis of the ratio of phosphorylated/total VEGFR-2 from three independent experiments ( n = 3).

    Article Snippet: The next day, pretreated the cells with vehicle or different doses of ABL, followed by exposure of some of the wells (n = 16) to VEGF-A (50 ng/mL) for 48 h. Cell growth was assessed using an MTT assay (Millipore Corporation, Temecula, CA, USA) according to the manufacturer’s protocol.

    Techniques: Western Blot

    ABL increases VEGF-induced HUVEC growth and proliferation. A) VEGF-induced EC growth monitored by the MTT assay following treatment with different doses of ABL or vehicle. The data represent the percent increase after 48 h relative to non-stimulated cells from 3 independent experiments. B) VEGF-induced [ 3 H]-thymidine incorporation following different doses of ABL or vehicle. The data represent the percent increase after 48 h relative to the non-stimulated cells from 3 independent experiments ( n = 3). * P

    Journal: PLoS ONE

    Article Title: Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells

    doi: 10.1371/journal.pone.0148968

    Figure Lengend Snippet: ABL increases VEGF-induced HUVEC growth and proliferation. A) VEGF-induced EC growth monitored by the MTT assay following treatment with different doses of ABL or vehicle. The data represent the percent increase after 48 h relative to non-stimulated cells from 3 independent experiments. B) VEGF-induced [ 3 H]-thymidine incorporation following different doses of ABL or vehicle. The data represent the percent increase after 48 h relative to the non-stimulated cells from 3 independent experiments ( n = 3). * P

    Article Snippet: The next day, pretreated the cells with vehicle or different doses of ABL, followed by exposure of some of the wells (n = 16) to VEGF-A (50 ng/mL) for 48 h. Cell growth was assessed using an MTT assay (Millipore Corporation, Temecula, CA, USA) according to the manufacturer’s protocol.

    Techniques: MTT Assay

    ABL enhances VEGFR-2 internalization in ECs. A) Colocalization of VEGFR-2 with endosome markers EEA1 in ECs. The ECs were pretreated with ABL for 2 h following fifteen minutes of VEGF stimulation, fixed, permeablized, and labeled with anti-VEGFR-2 (red) and anti-EEA1 (green) and processed for confocal microscopy. The endocytic trafficking of VEGFR2 without locating in endosome was observed in ECs (arrow). B) Colocalization of VEGFR-2 (red) and VE-cadherin (green) was observed in ECs (arrow). Nuclei were counterstained with DAPI (blue). Scale bar = 20 μm.

    Journal: PLoS ONE

    Article Title: Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells

    doi: 10.1371/journal.pone.0148968

    Figure Lengend Snippet: ABL enhances VEGFR-2 internalization in ECs. A) Colocalization of VEGFR-2 with endosome markers EEA1 in ECs. The ECs were pretreated with ABL for 2 h following fifteen minutes of VEGF stimulation, fixed, permeablized, and labeled with anti-VEGFR-2 (red) and anti-EEA1 (green) and processed for confocal microscopy. The endocytic trafficking of VEGFR2 without locating in endosome was observed in ECs (arrow). B) Colocalization of VEGFR-2 (red) and VE-cadherin (green) was observed in ECs (arrow). Nuclei were counterstained with DAPI (blue). Scale bar = 20 μm.

    Article Snippet: The next day, pretreated the cells with vehicle or different doses of ABL, followed by exposure of some of the wells (n = 16) to VEGF-A (50 ng/mL) for 48 h. Cell growth was assessed using an MTT assay (Millipore Corporation, Temecula, CA, USA) according to the manufacturer’s protocol.

    Techniques: Labeling, Confocal Microscopy

    ABL enhances Matrigel angiogenesis in vivo . A) Examples of CD31 immunofluorescence staining of Matrigel plugs containing VEGF-A or control buffer. B) The quantification data represent the percent increase in CD31-positive area in comparison with the control sample ( n = 6). C) Quantitative analysis of GAPDH-normalized VE-cadherin ( Cdh5 ) mRNA expression in Matrigel plugs containing VEGF-A or control buffer that were implanted in mice ( n = 3). Scale bars: 100 μm.

    Journal: PLoS ONE

    Article Title: Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells

    doi: 10.1371/journal.pone.0148968

    Figure Lengend Snippet: ABL enhances Matrigel angiogenesis in vivo . A) Examples of CD31 immunofluorescence staining of Matrigel plugs containing VEGF-A or control buffer. B) The quantification data represent the percent increase in CD31-positive area in comparison with the control sample ( n = 6). C) Quantitative analysis of GAPDH-normalized VE-cadherin ( Cdh5 ) mRNA expression in Matrigel plugs containing VEGF-A or control buffer that were implanted in mice ( n = 3). Scale bars: 100 μm.

    Article Snippet: The next day, pretreated the cells with vehicle or different doses of ABL, followed by exposure of some of the wells (n = 16) to VEGF-A (50 ng/mL) for 48 h. Cell growth was assessed using an MTT assay (Millipore Corporation, Temecula, CA, USA) according to the manufacturer’s protocol.

    Techniques: In Vivo, Immunofluorescence, Staining, Expressing, Mouse Assay

    Inhibition of VEGF-A-mediated PI3K/Akt pathway in HUVECs. The HUVECs were serum-starved for 24 h and stimulated in fresh medium with VEGF-A (25 ng/ml) and then treated with HY (0.062 μM) in combination with VEGF-A (25 ng/ml) for 24 h. The cells were exposed to a 585-nm LED light at a dose of 1.0 J/cm 2 and were incubation for 24 h. The cells were lysed and the proteins were harvested for western blot analysis of VEGF-A ( a ), p-Akt (Ser473) and Akt ( b ), and Bad ( c ). Densitometric measurements were analysed using AlphaEaseFC 4.0 software. The protein expression levels were normalized to those of the vehicle control (100%). Data are presented as means ± S.D. (n = 3); ** P

    Journal: Scientific Reports

    Article Title: Hypericin-photodynamic therapy induces human umbilical vein endothelial cell apoptosis

    doi: 10.1038/srep18398

    Figure Lengend Snippet: Inhibition of VEGF-A-mediated PI3K/Akt pathway in HUVECs. The HUVECs were serum-starved for 24 h and stimulated in fresh medium with VEGF-A (25 ng/ml) and then treated with HY (0.062 μM) in combination with VEGF-A (25 ng/ml) for 24 h. The cells were exposed to a 585-nm LED light at a dose of 1.0 J/cm 2 and were incubation for 24 h. The cells were lysed and the proteins were harvested for western blot analysis of VEGF-A ( a ), p-Akt (Ser473) and Akt ( b ), and Bad ( c ). Densitometric measurements were analysed using AlphaEaseFC 4.0 software. The protein expression levels were normalized to those of the vehicle control (100%). Data are presented as means ± S.D. (n = 3); ** P

    Article Snippet: The proteins were separated by 10% or 12% (for Bcl-2, Bax, cleaved caspase-9, procaspase-3, cleaved PARP, VEGF-A, phospho-Akt (Ser473, p-Akt), Akt, Bad and β-Actin) SDS-polyacrylamide gels and electrophoretically transferred onto PVDF membranes (Millipore, Massachusetts, USA).

    Techniques: Inhibition, Incubation, Western Blot, Software, Expressing

    VEGF siRNA-generating pDNA down regulates VEGF mRNA levels in rat cervix . The VEGF gene silencing efficiency of the selected siRNA-generating pDNA was tested in vivo in the cervix of pregnant rat from GD17–20. VEGF siRNA-generating pDNA was found to down-regulate VEGF compared to control. n = 5, ( p

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Delineation of VEGF-regulated genes and functions in the cervix of pregnant rodents by DNA microarray analysis

    doi: 10.1186/1477-7827-6-64

    Figure Lengend Snippet: VEGF siRNA-generating pDNA down regulates VEGF mRNA levels in rat cervix . The VEGF gene silencing efficiency of the selected siRNA-generating pDNA was tested in vivo in the cervix of pregnant rat from GD17–20. VEGF siRNA-generating pDNA was found to down-regulate VEGF compared to control. n = 5, ( p

    Article Snippet: Cervices were harvested, processed and evaluated for changes in levels of VEGF mRNA and whole genome gene expression using DNA microarray data, as described below. b) Pregnant mice and ovariectomized non-pregnant rats and mice treated with either mouse recombinant VEGF protein (10 ng/mouse recombinant VEGF; Calbiochem, La Jolla, CA, once daily from GD13–17, intra-vaginally), VEGF receptor antagonist (as described earlier, but treated daily), or vehicle only, were utilized to confirm the DNA microarray data above using real-time PCR, and for morphological studies (SEM), described below.

    Techniques: In Vivo

    VEGF siRNA-generating pDNA down regulates VEGF mRNA levels in rat heart fibroblasts . The VEGF gene silencing efficiency by the three siRNA-generating pDNAs were tested in vitro using rat heart fibroblast primary cell transfection. VEGF siRNA-generating pDNA was found to down-regulate VEGF in cultured rat heart fibroblasts compared to control. The pDNA with the best silencing efficiency was selected and used in the rat cervix to test its efficiency to down-regulate local cervical VEGF in vivo (see Figure 2). n = 5, ( p

    Journal: Reproductive Biology and Endocrinology : RB & E

    Article Title: Delineation of VEGF-regulated genes and functions in the cervix of pregnant rodents by DNA microarray analysis

    doi: 10.1186/1477-7827-6-64

    Figure Lengend Snippet: VEGF siRNA-generating pDNA down regulates VEGF mRNA levels in rat heart fibroblasts . The VEGF gene silencing efficiency by the three siRNA-generating pDNAs were tested in vitro using rat heart fibroblast primary cell transfection. VEGF siRNA-generating pDNA was found to down-regulate VEGF in cultured rat heart fibroblasts compared to control. The pDNA with the best silencing efficiency was selected and used in the rat cervix to test its efficiency to down-regulate local cervical VEGF in vivo (see Figure 2). n = 5, ( p

    Article Snippet: Cervices were harvested, processed and evaluated for changes in levels of VEGF mRNA and whole genome gene expression using DNA microarray data, as described below. b) Pregnant mice and ovariectomized non-pregnant rats and mice treated with either mouse recombinant VEGF protein (10 ng/mouse recombinant VEGF; Calbiochem, La Jolla, CA, once daily from GD13–17, intra-vaginally), VEGF receptor antagonist (as described earlier, but treated daily), or vehicle only, were utilized to confirm the DNA microarray data above using real-time PCR, and for morphological studies (SEM), described below.

    Techniques: In Vitro, Transfection, Cell Culture, In Vivo