recombinant human vegf a  (R&D Systems)

 
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    Name:
    Recombinant Human VEGF Ax Protein
    Description:
    The Recombinant Human VEGF Ax Protein from R D Systems is derived from CHO The Recombinant Human VEGF Ax Protein has been validated for the following applications Bioactivity
    Catalog Number:
    9018-VE-025
    Price:
    329
    Category:
    Proteins and Enzymes
    Source:
    CHO-derived Recombinant Human VEGF-Ax Protein
    Applications:
    Bioactivity
    Purity:
    >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie« Blue Staining.
    Conjugate:
    Unconjugated
    Size:
    25 ug
    Buy from Supplier


    Structured Review

    R&D Systems recombinant human vegf a
    uPARAP deficiency stimulates <t>VEGF-C-driven</t> lymphangiogenesis. a Schematic of VEGF ligand interaction with VEGFR-2 and VEGFR-3 homodimers or VEGFR-2/VEGFR-3 heterodimers. b – e Gelatin sponges soaked with PBS ( b ), VEGF-C ( c ), <t>VEGF-A</t> ( d ), or mutated VEGF-C Cys156Ser ( e ) were implanted in mouse ears. White dots delineate the sponge in the ear. Lymphatic vasculature was examined by LYVE-1 (green) immunostaining. Histograms represent the area density of vessels quantified by a computer-assisted method and expressed as percentage of WT control ( b – d n = 6; e n = 8). Bars = 500 µm. f Indocyanin Green (ICG) clearance in VEGF-C-soaked sponges in uPARAP WT and KO mice using Xenogen IVIS. Histogram represents mean fluorescence signal detected at each time point ( n = 5). All results are expressed as mean ± SEM, and statistical analyses were performed using a non-parametric Mann–Whitney test. * P
    The Recombinant Human VEGF Ax Protein from R D Systems is derived from CHO The Recombinant Human VEGF Ax Protein has been validated for the following applications Bioactivity
    https://www.bioz.com/result/recombinant human vegf a/product/R&D Systems
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant human vegf a - by Bioz Stars, 2021-03
    94/100 stars

    Images

    1) Product Images from "uPARAP/Endo180 receptor is a gatekeeper of VEGFR-2/VEGFR-3 heterodimerisation during pathological lymphangiogenesis"

    Article Title: uPARAP/Endo180 receptor is a gatekeeper of VEGFR-2/VEGFR-3 heterodimerisation during pathological lymphangiogenesis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07514-1

    uPARAP deficiency stimulates VEGF-C-driven lymphangiogenesis. a Schematic of VEGF ligand interaction with VEGFR-2 and VEGFR-3 homodimers or VEGFR-2/VEGFR-3 heterodimers. b – e Gelatin sponges soaked with PBS ( b ), VEGF-C ( c ), VEGF-A ( d ), or mutated VEGF-C Cys156Ser ( e ) were implanted in mouse ears. White dots delineate the sponge in the ear. Lymphatic vasculature was examined by LYVE-1 (green) immunostaining. Histograms represent the area density of vessels quantified by a computer-assisted method and expressed as percentage of WT control ( b – d n = 6; e n = 8). Bars = 500 µm. f Indocyanin Green (ICG) clearance in VEGF-C-soaked sponges in uPARAP WT and KO mice using Xenogen IVIS. Histogram represents mean fluorescence signal detected at each time point ( n = 5). All results are expressed as mean ± SEM, and statistical analyses were performed using a non-parametric Mann–Whitney test. * P
    Figure Legend Snippet: uPARAP deficiency stimulates VEGF-C-driven lymphangiogenesis. a Schematic of VEGF ligand interaction with VEGFR-2 and VEGFR-3 homodimers or VEGFR-2/VEGFR-3 heterodimers. b – e Gelatin sponges soaked with PBS ( b ), VEGF-C ( c ), VEGF-A ( d ), or mutated VEGF-C Cys156Ser ( e ) were implanted in mouse ears. White dots delineate the sponge in the ear. Lymphatic vasculature was examined by LYVE-1 (green) immunostaining. Histograms represent the area density of vessels quantified by a computer-assisted method and expressed as percentage of WT control ( b – d n = 6; e n = 8). Bars = 500 µm. f Indocyanin Green (ICG) clearance in VEGF-C-soaked sponges in uPARAP WT and KO mice using Xenogen IVIS. Histogram represents mean fluorescence signal detected at each time point ( n = 5). All results are expressed as mean ± SEM, and statistical analyses were performed using a non-parametric Mann–Whitney test. * P

    Techniques Used: Immunostaining, Mouse Assay, Fluorescence, MANN-WHITNEY

    uPARAP downregulation impairs LEC chemotactic migration toward a VEGF-C gradient. LECs were transfected with a siRNA targeting uPARAP (siU1 and siU2) or a control siRNA (Ctr). RT-PCR ( a ) and Western blot ( b ) analyses of uPARAP, VEGFR-3 and VEGFR-2 expression (28S and GAPDH = internal controls). c Proliferation upon VEGF-C or VEGF-A stimulation. Data are those of one representative assay out of three ( n = 12 for T0 and n = 8 for T48 h, biological replicates). d Scratch assay in the presence of control medium ( n = 10), VEGF-C ( n = 10) or VEGF-A ( n = 3) (biological replicates in all conditions). e Phalloidin and uPARAP stainings of LECs migrating toward a VEGF-C or VEGF-A gradient in Ibidi µ-slides. Bars = 25 µm in left images and 50 µm in right images (higher magnification of inserts). f Boyden Chamber assay in the presence of control medium, wild type VEGF-C, VEGF-A, or mutated VEGF-C Cys156Ser . Histograms correspond to the mean ( n = 6 for control medium, VEGF-C and VEGF-A, and n = 3 for mutated VEGF-C Cys156Ser , biological replicates) ± SEM. g Chemotactic response to a gradient of complete medium, VEGF-C or VEGF-A. The rose diagram represents the direction of migration of more than 30 cells. Black arrows indicate the mean migration direction. The absence of arrow in VEGF-C diagrams reflects impaired directionality upon uPARAP silencing. Histograms represent the migratory speed (µm/min) where results are expressed as mean ( n = 6 biological replicates for complete medium and VEGF-C; n = 3 biological replicates for VEGF-A) ± SEM. Statistical analyses were performed using a non-parametric Mann–Whitney test. * P
    Figure Legend Snippet: uPARAP downregulation impairs LEC chemotactic migration toward a VEGF-C gradient. LECs were transfected with a siRNA targeting uPARAP (siU1 and siU2) or a control siRNA (Ctr). RT-PCR ( a ) and Western blot ( b ) analyses of uPARAP, VEGFR-3 and VEGFR-2 expression (28S and GAPDH = internal controls). c Proliferation upon VEGF-C or VEGF-A stimulation. Data are those of one representative assay out of three ( n = 12 for T0 and n = 8 for T48 h, biological replicates). d Scratch assay in the presence of control medium ( n = 10), VEGF-C ( n = 10) or VEGF-A ( n = 3) (biological replicates in all conditions). e Phalloidin and uPARAP stainings of LECs migrating toward a VEGF-C or VEGF-A gradient in Ibidi µ-slides. Bars = 25 µm in left images and 50 µm in right images (higher magnification of inserts). f Boyden Chamber assay in the presence of control medium, wild type VEGF-C, VEGF-A, or mutated VEGF-C Cys156Ser . Histograms correspond to the mean ( n = 6 for control medium, VEGF-C and VEGF-A, and n = 3 for mutated VEGF-C Cys156Ser , biological replicates) ± SEM. g Chemotactic response to a gradient of complete medium, VEGF-C or VEGF-A. The rose diagram represents the direction of migration of more than 30 cells. Black arrows indicate the mean migration direction. The absence of arrow in VEGF-C diagrams reflects impaired directionality upon uPARAP silencing. Histograms represent the migratory speed (µm/min) where results are expressed as mean ( n = 6 biological replicates for complete medium and VEGF-C; n = 3 biological replicates for VEGF-A) ± SEM. Statistical analyses were performed using a non-parametric Mann–Whitney test. * P

    Techniques Used: Migration, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Wound Healing Assay, Boyden Chamber Assay, MANN-WHITNEY

    2) Product Images from "The Effect of Interleukin 38 on Angiogenesis in a Model of Oxygen-induced Retinopathy"

    Article Title: The Effect of Interleukin 38 on Angiogenesis in a Model of Oxygen-induced Retinopathy

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-03079-z

    IL-38 attenuates endothelial cell proliferation. HRECs (1 × 10 4 ) were cultured with VEGF/IL-38/anti-IL-38/IgG at 37 °C in a CO 2 incubator and the proliferation was determined by MTT method. Shown are results from one representative experiment of three performed (in triplicates). * P
    Figure Legend Snippet: IL-38 attenuates endothelial cell proliferation. HRECs (1 × 10 4 ) were cultured with VEGF/IL-38/anti-IL-38/IgG at 37 °C in a CO 2 incubator and the proliferation was determined by MTT method. Shown are results from one representative experiment of three performed (in triplicates). * P

    Techniques Used: Cell Culture, MTT Assay

    IL-38 reduces endothelial cell tube formation. ( a ) 40,000 HRECs/well were seeded on Matrigel containing VEGF/IL-38/anti-IL-38/IgG in depleted medium. The cells were cultured for 18 h at 37 °C 5% CO 2 . Tube formation was quantified by counting the tube-like structures in the gel and data were presented as the number of branches per field. Scale bar, 200 μm. ( b ) Total length of tubule structure were quantified. * P
    Figure Legend Snippet: IL-38 reduces endothelial cell tube formation. ( a ) 40,000 HRECs/well were seeded on Matrigel containing VEGF/IL-38/anti-IL-38/IgG in depleted medium. The cells were cultured for 18 h at 37 °C 5% CO 2 . Tube formation was quantified by counting the tube-like structures in the gel and data were presented as the number of branches per field. Scale bar, 200 μm. ( b ) Total length of tubule structure were quantified. * P

    Techniques Used: Cell Culture

    3) Product Images from "Cytomegalovirus Impairs Cytotrophoblast-Induced Lymphangiogenesis and Vascular Remodeling in an in Vivo Human Placentation Model"

    Article Title: Cytomegalovirus Impairs Cytotrophoblast-Induced Lymphangiogenesis and Vascular Remodeling in an in Vivo Human Placentation Model

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2012.08.003

    Quantification of cytokines from AD169- and VR1814-infected cytotrophoblasts and placental villous explants in vitro . Primary cytotrophoblasts ( A , C , E , G , and I ) and placental villous explants ( B , D , F , H , and J ) are infected after 12 hours on Matrigel. Conditioned medium is removed at 1 and 3 days (d) after infection, and factors are quantified using ELISA: VEGF-C, bFGF, sVEGFR-3, bioactive VEGF-A, and IL-6. Results are from three to six independent experiments. Cont., control.
    Figure Legend Snippet: Quantification of cytokines from AD169- and VR1814-infected cytotrophoblasts and placental villous explants in vitro . Primary cytotrophoblasts ( A , C , E , G , and I ) and placental villous explants ( B , D , F , H , and J ) are infected after 12 hours on Matrigel. Conditioned medium is removed at 1 and 3 days (d) after infection, and factors are quantified using ELISA: VEGF-C, bFGF, sVEGFR-3, bioactive VEGF-A, and IL-6. Results are from three to six independent experiments. Cont., control.

    Techniques Used: Infection, In Vitro, Enzyme-linked Immunosorbent Assay

    Aberrant lymphangiogenesis in VR1814-infected human placental villi in vivo . LYVE-1–immunostained lymphatic vessels and endothelial cells are shown. A and B: Normal vessels in mock-infected controls. C – E: Aberrant vessels in VR1814-infected placental implants. F – H: Co-immunostaining for LYVE-1 and podoplanin in VR1814-infected placental implants. I and J: Expression of LYVE-1 and integrin α9β1 in adjacent sections of a VR1814-infected placental implant. K: Ki-67 immunostains proliferating LYVE-1–expressing lymphatic endothelial cells. L: Proliferation index of lymphatic endothelial cells in mock-infected controls and infected villi. The numbers of LYVE-1– and Ki-67–positive lymphatic endothelial cells per field are counted. The proliferation index is expressed as the percentage of LYVE-1–positive cells that are also Ki-67 positive. Control implants ( n = 3, with 15 fields counted) and VR1814-infected implants ( n = 9, with 27 fields counted) are shown. * P = 0.003 (Student's t -test). M and N: Immunostaining for VEGF-A ( M ) and VEGF-C ( N ) in implants. Scale bars: 20 μm ( A – J ); 5 μm ( K ); 5 μm ( M and N ). CTB, cytotrophoblast.
    Figure Legend Snippet: Aberrant lymphangiogenesis in VR1814-infected human placental villi in vivo . LYVE-1–immunostained lymphatic vessels and endothelial cells are shown. A and B: Normal vessels in mock-infected controls. C – E: Aberrant vessels in VR1814-infected placental implants. F – H: Co-immunostaining for LYVE-1 and podoplanin in VR1814-infected placental implants. I and J: Expression of LYVE-1 and integrin α9β1 in adjacent sections of a VR1814-infected placental implant. K: Ki-67 immunostains proliferating LYVE-1–expressing lymphatic endothelial cells. L: Proliferation index of lymphatic endothelial cells in mock-infected controls and infected villi. The numbers of LYVE-1– and Ki-67–positive lymphatic endothelial cells per field are counted. The proliferation index is expressed as the percentage of LYVE-1–positive cells that are also Ki-67 positive. Control implants ( n = 3, with 15 fields counted) and VR1814-infected implants ( n = 9, with 27 fields counted) are shown. * P = 0.003 (Student's t -test). M and N: Immunostaining for VEGF-A ( M ) and VEGF-C ( N ) in implants. Scale bars: 20 μm ( A – J ); 5 μm ( K ); 5 μm ( M and N ). CTB, cytotrophoblast.

    Techniques Used: Infection, In Vivo, Immunostaining, Expressing, Positive Control, CtB Assay

    4) Product Images from "Comparative Phosphoproteomics Analysis of VEGF and Angiopoietin-1 Signaling Reveals ZO-1 as a Critical Regulator of Endothelial Cell Proliferation *"

    Article Title: Comparative Phosphoproteomics Analysis of VEGF and Angiopoietin-1 Signaling Reveals ZO-1 as a Critical Regulator of Endothelial Cell Proliferation *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M115.053298

    Comparative analysis of the phosphoproteomes of VEGF and Ang-1 treated cells. A, A hierarchical clustering heatmap showing three different clusters of biological processes enrichment of VEGF or Ang-1 regulated phosphoproteins. Phosphoproteins regulated by VEGF or Ang-1 were analyzed using the GO biological process annotation built in STRING database. Red corresponds to GO terms significantly enriched ( p value
    Figure Legend Snippet: Comparative analysis of the phosphoproteomes of VEGF and Ang-1 treated cells. A, A hierarchical clustering heatmap showing three different clusters of biological processes enrichment of VEGF or Ang-1 regulated phosphoproteins. Phosphoproteins regulated by VEGF or Ang-1 were analyzed using the GO biological process annotation built in STRING database. Red corresponds to GO terms significantly enriched ( p value

    Techniques Used:

    Analysis of the phosphoproteome in VEGF or Ang-1 treated BAECs. A , Activation of Akt, MAPK and eNOS, using the indicated phosphospecific antibodies, was monitored in BAECs treated with VEGF (40 ng/ml) or Ang-1 (100 ng/ml) for the indicated times. Equal protein loading was confirmed by reprobing the membranes with antibodies against total Akt, MAPK and eNOS. These experiments were repeated at least three times with similar results. B , Volcano plot showing the distribution of phosphopeptides abundance of VEGF or Ang-1 over control (fold-change, x axis) as a function of statistical significance (-log10 of p value, y axis). Red circles correspond to 255 significantly up-regulated peptides and 9 significantly down-regulated peptides upon VEGF stimulation. Green squares correspond to 248 significantly up-regulated peptides upon Ang-1 stimulation and 11 significantly down-regulated peptides to Ang-1 treatment. A cut-off of log2 fold-change > 0.5 and log2 fold-change
    Figure Legend Snippet: Analysis of the phosphoproteome in VEGF or Ang-1 treated BAECs. A , Activation of Akt, MAPK and eNOS, using the indicated phosphospecific antibodies, was monitored in BAECs treated with VEGF (40 ng/ml) or Ang-1 (100 ng/ml) for the indicated times. Equal protein loading was confirmed by reprobing the membranes with antibodies against total Akt, MAPK and eNOS. These experiments were repeated at least three times with similar results. B , Volcano plot showing the distribution of phosphopeptides abundance of VEGF or Ang-1 over control (fold-change, x axis) as a function of statistical significance (-log10 of p value, y axis). Red circles correspond to 255 significantly up-regulated peptides and 9 significantly down-regulated peptides upon VEGF stimulation. Green squares correspond to 248 significantly up-regulated peptides upon Ang-1 stimulation and 11 significantly down-regulated peptides to Ang-1 treatment. A cut-off of log2 fold-change > 0.5 and log2 fold-change

    Techniques Used: Activation Assay

    Phosphoprotein interaction network of VEGF and Ang-1 treated cells. Protein interaction networks generated with VEGF ( A ) and Ang-1 ( B ) regulated phosphoproteins using STRING database version 9.1 with medium confidence and visualized by Cytoscape. The proteins that did not interact with any other proteins are not shown. The interaction network was subjected to a cluster analysis using the algorithm MCODE based on the protein interaction level. Clusters are represented with different colors. Phosphoproteins in white are not part of any cluster. The tables contain the gene names and the functional enrichment of VEGF ( C ) and Ang-1 ( D ) clusters. The functional enrichment was manually annotated using GO terms of the biological process category and Uniprot database. The green cluster shows the junctional proteins in the same cluster with MAPK1 in VEGF but not in Ang-1 interaction network.
    Figure Legend Snippet: Phosphoprotein interaction network of VEGF and Ang-1 treated cells. Protein interaction networks generated with VEGF ( A ) and Ang-1 ( B ) regulated phosphoproteins using STRING database version 9.1 with medium confidence and visualized by Cytoscape. The proteins that did not interact with any other proteins are not shown. The interaction network was subjected to a cluster analysis using the algorithm MCODE based on the protein interaction level. Clusters are represented with different colors. Phosphoproteins in white are not part of any cluster. The tables contain the gene names and the functional enrichment of VEGF ( C ) and Ang-1 ( D ) clusters. The functional enrichment was manually annotated using GO terms of the biological process category and Uniprot database. The green cluster shows the junctional proteins in the same cluster with MAPK1 in VEGF but not in Ang-1 interaction network.

    Techniques Used: Generated, Functional Assay

    5) Product Images from "A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity"

    Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7601774

    The 3′UTR of VEGF-A mRNA mediates translation inhibition. ( A ) Schematic of VEGF-A mRNA and chimeric luciferase constructs used for in vitro translation (top panel). The m 7 G cap is indicated by an open circle, the IRES by a light gray rectangle, the putative GAIT element by a black rectangle, and the AREs by dark gray rectangles. Capped, FLuc-VEGF-A 3′UTR (11–900) -A 30 RNA was translated in RRL containing [ 35 S]Met, and in absence or presence of cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h (middle panel). Capped, RLuc RNA lacking the GAIT element was co-translated in each reaction as control. Translation reactions were resolved on SDS–10% polyacrylamide gel. The same RNAs were translated in the presence of cytosolic extract from 24-h, IFN-γ-treated U937 cells, and in the presence of 10- and 50-fold molar excess of in vitro transcribed VEGF-A 3′UTR RNA as competitor (bottom panel). ( B ) Schematic of chimeric luciferase constructs used for in vitro translation (top panel). In vitro translation, in presence of IFN-γ-treated U937 cytosolic extracts, of capped FLuc-VEGF-A 3′UTR (324–455) -A 30 encompassing the putative GAIT element (middle panel), and FLuc-VEGF-A 3′UTR (441–560) -A 30 (bottom panel). RLuc RNA was co-translated in each reaction.
    Figure Legend Snippet: The 3′UTR of VEGF-A mRNA mediates translation inhibition. ( A ) Schematic of VEGF-A mRNA and chimeric luciferase constructs used for in vitro translation (top panel). The m 7 G cap is indicated by an open circle, the IRES by a light gray rectangle, the putative GAIT element by a black rectangle, and the AREs by dark gray rectangles. Capped, FLuc-VEGF-A 3′UTR (11–900) -A 30 RNA was translated in RRL containing [ 35 S]Met, and in absence or presence of cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h (middle panel). Capped, RLuc RNA lacking the GAIT element was co-translated in each reaction as control. Translation reactions were resolved on SDS–10% polyacrylamide gel. The same RNAs were translated in the presence of cytosolic extract from 24-h, IFN-γ-treated U937 cells, and in the presence of 10- and 50-fold molar excess of in vitro transcribed VEGF-A 3′UTR RNA as competitor (bottom panel). ( B ) Schematic of chimeric luciferase constructs used for in vitro translation (top panel). In vitro translation, in presence of IFN-γ-treated U937 cytosolic extracts, of capped FLuc-VEGF-A 3′UTR (324–455) -A 30 encompassing the putative GAIT element (middle panel), and FLuc-VEGF-A 3′UTR (441–560) -A 30 (bottom panel). RLuc RNA was co-translated in each reaction.

    Techniques Used: Inhibition, Luciferase, Construct, In Vitro

    Translational silencing of VEGF-A expression in vivo. ( A ) RT–PCR analysis of total RNA from U937 cells treated with IFN-γ for 0, 8, or 24 h. RT–PCR was done using primers specific for VEGF-A (top panel) and GAPDH (bottom panel). Real-time PCR results indicating the increase in VEGF-A mRNA expression in IFN-γ-treated cells compared to untreated cells are included below the top panel (expressed as fold-increase normalized to β-actin). ( B ) Cell lysates from U937 cells treated with IFN-γ for 0, 8, or 24 h were processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top) and anti-GAPDH (bottom panel) antibodies. ( C ) RT–PCR analysis of total RNA from human PBMCs treated with IFN-γ for 0, 8, or 24 h. RT–PCR was performed using primers specific for VEGF-A (top panel) and GAPDH (bottom panel). ( D ) Cell lysates from PBMCs treated with IFN-γ for 0, 8, or 24 h were processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top) and anti-GAPDH (bottom panel) antibodies. ( E ) U937 cells were treated with IFN-γ for up to 24 h. At the end of each interval, cells were metabolically labeled with [ 35 S]Met/Cys for 1 h. Conditioned media and cell lysates were immunoprecipitated with anti-VEGF-A antibody and resolved by electrophoresis on SDS–10% polyacrylamide gel (top panel). Monomeric and dimeric VEGF-A forms are indicated by arrows. The same samples were subjected to electrophoresis without immunoprecipitation (bottom). ( F ) U937 cells were treated with IFN-γ for 8 or 24 h and cytosolic extracts were fractionated into polysomal and non-polysomal, RNP fractions by ultracentrifugation on a 20% sucrose cushion in the presence or absence of 10 mM EDTA. RNA associated with each fraction was isolated and subjected to RT–PCR using primers specific for VEGF-A (top panel) and GAPDH (bottom panel).
    Figure Legend Snippet: Translational silencing of VEGF-A expression in vivo. ( A ) RT–PCR analysis of total RNA from U937 cells treated with IFN-γ for 0, 8, or 24 h. RT–PCR was done using primers specific for VEGF-A (top panel) and GAPDH (bottom panel). Real-time PCR results indicating the increase in VEGF-A mRNA expression in IFN-γ-treated cells compared to untreated cells are included below the top panel (expressed as fold-increase normalized to β-actin). ( B ) Cell lysates from U937 cells treated with IFN-γ for 0, 8, or 24 h were processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top) and anti-GAPDH (bottom panel) antibodies. ( C ) RT–PCR analysis of total RNA from human PBMCs treated with IFN-γ for 0, 8, or 24 h. RT–PCR was performed using primers specific for VEGF-A (top panel) and GAPDH (bottom panel). ( D ) Cell lysates from PBMCs treated with IFN-γ for 0, 8, or 24 h were processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top) and anti-GAPDH (bottom panel) antibodies. ( E ) U937 cells were treated with IFN-γ for up to 24 h. At the end of each interval, cells were metabolically labeled with [ 35 S]Met/Cys for 1 h. Conditioned media and cell lysates were immunoprecipitated with anti-VEGF-A antibody and resolved by electrophoresis on SDS–10% polyacrylamide gel (top panel). Monomeric and dimeric VEGF-A forms are indicated by arrows. The same samples were subjected to electrophoresis without immunoprecipitation (bottom). ( F ) U937 cells were treated with IFN-γ for 8 or 24 h and cytosolic extracts were fractionated into polysomal and non-polysomal, RNP fractions by ultracentrifugation on a 20% sucrose cushion in the presence or absence of 10 mM EDTA. RNA associated with each fraction was isolated and subjected to RT–PCR using primers specific for VEGF-A (top panel) and GAPDH (bottom panel).

    Techniques Used: Expressing, In Vivo, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Metabolic Labelling, Labeling, Immunoprecipitation, Electrophoresis, Isolation

    The GAIT complex binds the VEGF-A GAIT element and causes translational silencing. ( A ) RNA EMSA using 32 P-labeled Cp and VEGF-A GAIT element probes. The riboprobes were incubated with cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h. RNA–protein complexes were resolved by electrophoresis on a nondenaturing 5% polyacrylamide gel. ( B ) RNA–protein complexes formed between 32 P-labeled VEGF-A GAIT element RNA and lysates from 24-h, IFN-γ-treated U937 cells were supershifted with antibodies against GAIT complex components. The cell lysate was incubated with the respective antibodies or non-immune IgG before incubation with the riboprobe. ( C ) Lysate from U937 cells treated with IFN-γ for 24 h was incubated with protein-A Sepharose beads coupled to anti-EPRS antibody (or to pre-immune serum, Pre-im.) to immunodeplete the GAIT complex. The beads were pelleted, and the supernatant subjected to immunoblotting with anti-EPRS antibody to verify effective immunodepletion. ( D ) At 24-h, IFN-γ-treated U937 cell lysates, immunodepleted with anti-EPRS antibody or pre-immune serum, were added to in vitro translation reactions containing FLuc-VEGF-A 3′UTR (11–900) -A 30 and RLuc RNAs.
    Figure Legend Snippet: The GAIT complex binds the VEGF-A GAIT element and causes translational silencing. ( A ) RNA EMSA using 32 P-labeled Cp and VEGF-A GAIT element probes. The riboprobes were incubated with cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h. RNA–protein complexes were resolved by electrophoresis on a nondenaturing 5% polyacrylamide gel. ( B ) RNA–protein complexes formed between 32 P-labeled VEGF-A GAIT element RNA and lysates from 24-h, IFN-γ-treated U937 cells were supershifted with antibodies against GAIT complex components. The cell lysate was incubated with the respective antibodies or non-immune IgG before incubation with the riboprobe. ( C ) Lysate from U937 cells treated with IFN-γ for 24 h was incubated with protein-A Sepharose beads coupled to anti-EPRS antibody (or to pre-immune serum, Pre-im.) to immunodeplete the GAIT complex. The beads were pelleted, and the supernatant subjected to immunoblotting with anti-EPRS antibody to verify effective immunodepletion. ( D ) At 24-h, IFN-γ-treated U937 cell lysates, immunodepleted with anti-EPRS antibody or pre-immune serum, were added to in vitro translation reactions containing FLuc-VEGF-A 3′UTR (11–900) -A 30 and RLuc RNAs.

    Techniques Used: Labeling, Incubation, Electrophoresis, In Vitro

    Functional identification of the VEGF-A 3′UTR GAIT element. ( A ) Folding structures of the Cp (nt 78–106) and the putative VEGF-A GAIT (nt 358–386) elements as predicted by the Mfold algorithm. Base pairing between A7:U23 and U8:A22 was disallowed while folding the VEGF-A GAIT element. ( B ) Chimeric luciferase constructs containing wild-type or mutant VEGF-A 3′UTR GAIT elements. Capped and poly-A tailed RNAs, containing the putative VEGF-A GAIT element (FLuc-VEGF-A GAIT-A 30 ) or a mutant (U10C) GAIT element (FLuc-VEGF-A GAIT mut -A 30 ), downstream of FLuc (top panel), were subjected to in vitro translation in presence of cytosolic extracts from IFN-γ-treated U937 cells (bottom panel). RLuc RNA was co-translated in each reaction. ( C ) Capped and poly-A tailed RNAs, containing the putative VEGF-A GAIT element or a mutant GAIT element as in (B), were subjected to in vitro translation in presence of cytosolic extracts from IFN-γ-treated human PBMC (top panel). RLuc RNA was co-translated in each reaction. Fluc was quantified by densitometry, normalized to Rluc, and expressed as per cent of control condition without cell lysate (bottom). ( D ) U937 cells were transfected with eukaryotic, CMV-driven expression vectors containing the FLuc gene upstream of either wild-type (CMV-FLuc-VEGF-A GAIT-A 30 ) or mutant VEGF-A GAIT element (CMV-FLuc-VEGF-A GAIT mut -A 30 ) or lacking any GAIT element (CMV-FLuc). Cells were co-transfected with a vector containing RLuc gene under the SV40 promoter. Following transfection, cells were treated with IFN-γ for 8 (gray bars) or 24 h (black bars), or with medium alone (hatched bars). Luciferase activity in cell lysates was measured by dual luciferase assay. Results show mean and standard deviation of values from three independent experiments.
    Figure Legend Snippet: Functional identification of the VEGF-A 3′UTR GAIT element. ( A ) Folding structures of the Cp (nt 78–106) and the putative VEGF-A GAIT (nt 358–386) elements as predicted by the Mfold algorithm. Base pairing between A7:U23 and U8:A22 was disallowed while folding the VEGF-A GAIT element. ( B ) Chimeric luciferase constructs containing wild-type or mutant VEGF-A 3′UTR GAIT elements. Capped and poly-A tailed RNAs, containing the putative VEGF-A GAIT element (FLuc-VEGF-A GAIT-A 30 ) or a mutant (U10C) GAIT element (FLuc-VEGF-A GAIT mut -A 30 ), downstream of FLuc (top panel), were subjected to in vitro translation in presence of cytosolic extracts from IFN-γ-treated U937 cells (bottom panel). RLuc RNA was co-translated in each reaction. ( C ) Capped and poly-A tailed RNAs, containing the putative VEGF-A GAIT element or a mutant GAIT element as in (B), were subjected to in vitro translation in presence of cytosolic extracts from IFN-γ-treated human PBMC (top panel). RLuc RNA was co-translated in each reaction. Fluc was quantified by densitometry, normalized to Rluc, and expressed as per cent of control condition without cell lysate (bottom). ( D ) U937 cells were transfected with eukaryotic, CMV-driven expression vectors containing the FLuc gene upstream of either wild-type (CMV-FLuc-VEGF-A GAIT-A 30 ) or mutant VEGF-A GAIT element (CMV-FLuc-VEGF-A GAIT mut -A 30 ) or lacking any GAIT element (CMV-FLuc). Cells were co-transfected with a vector containing RLuc gene under the SV40 promoter. Following transfection, cells were treated with IFN-γ for 8 (gray bars) or 24 h (black bars), or with medium alone (hatched bars). Luciferase activity in cell lysates was measured by dual luciferase assay. Results show mean and standard deviation of values from three independent experiments.

    Techniques Used: Functional Assay, Luciferase, Construct, Mutagenesis, In Vitro, Transfection, Expressing, Plasmid Preparation, Activity Assay, Standard Deviation

    Ablation of the GAIT complex in vivo prevents translational silencing of VEGF-A. ( A ) Lysates from U937 cells stably transfected with pSUPER vector (U937-pSUPER) or pSUPER encoding a short hairpin RNA targeting L13a (U937-L13a-SHR) were immunoblotted with anti-L13a antibody. ( B ) Lysates from the stably transfected cell lines in (A) were treated with IFN-γ for 0, 8, or 24 h and processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top panel) and anti-GAPDH (bottom panel) antibodies. ( C ) Total RNA was isolated from the stably transfected cell lines treated with IFN-γ for 0, 8, or 24 h, and analyzed by RT–PCR using primers specific for VEGF-A (top panel) and β-actin (bottom panel). Real-time PCR results indicating increased VEGF-A mRNA expression in IFN-γ-treated cells compared to untreated cells (expressed as fold-increase normalized to β-actin) are inserted below the top panel. ( D ) The cell lines described in (A) were treated with IFN-γ for 24 h and lysates immunoprecipitated with anti-EPRS antibody, followed by RT–PCR with VEGF-A-specific primers.
    Figure Legend Snippet: Ablation of the GAIT complex in vivo prevents translational silencing of VEGF-A. ( A ) Lysates from U937 cells stably transfected with pSUPER vector (U937-pSUPER) or pSUPER encoding a short hairpin RNA targeting L13a (U937-L13a-SHR) were immunoblotted with anti-L13a antibody. ( B ) Lysates from the stably transfected cell lines in (A) were treated with IFN-γ for 0, 8, or 24 h and processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top panel) and anti-GAPDH (bottom panel) antibodies. ( C ) Total RNA was isolated from the stably transfected cell lines treated with IFN-γ for 0, 8, or 24 h, and analyzed by RT–PCR using primers specific for VEGF-A (top panel) and β-actin (bottom panel). Real-time PCR results indicating increased VEGF-A mRNA expression in IFN-γ-treated cells compared to untreated cells (expressed as fold-increase normalized to β-actin) are inserted below the top panel. ( D ) The cell lines described in (A) were treated with IFN-γ for 24 h and lysates immunoprecipitated with anti-EPRS antibody, followed by RT–PCR with VEGF-A-specific primers.

    Techniques Used: In Vivo, Stable Transfection, Transfection, Plasmid Preparation, shRNA, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, Immunoprecipitation

    VEGF-A mRNA interacts with the GAIT complex. ( A ) Secondary structure and sequence features of the human Cp GAIT element (top panel). The query pattern, based on the secondary structure and sequence features of the Cp GAIT element, was used to search a nonredundant 3′UTR database using the PatSearch program (bottom panel). Following the syntax of the PatSearch algorithm, allowed base-pairs are represented by r number and patterns defined by p number. The GAIT element-specific stems and loops are shown below. ( B ) PatSearch result predicted the presence of GAIT elements in Cp and VEGF-A 3′UTR. UTRdb ID refers to the sequence entry in the UTR database, and sequence position refers to the 3′UTR position of the sequence encoding the predicted GAIT element. ( C ) To show VEGF-A mRNA interaction with the GAIT complex in vivo , U937 cells were treated with IFN-γ for 8 or 24 h, and lysates were immunoprecipitated (IP) with anti-EPRS antibody to isolate GAIT complex, or with control pre-immune (Pre-im.) serum. RNA associated with the GAIT complex, or present in the non-immunoprecipitated supernatant (Sup.), was subjected to RT–PCR using primers specific for VEGF-A or β-actin mRNA, and products were resolved in 1.6% agarose gels. ( D ) To verify antibody specificity, lysate from U937 cells treated with IFN-γ for 24 h was immunoprecipitated with polyclonal anti-human EPRS antibody and immunoblotted with the same antibody, or with pre-immune serum as control.
    Figure Legend Snippet: VEGF-A mRNA interacts with the GAIT complex. ( A ) Secondary structure and sequence features of the human Cp GAIT element (top panel). The query pattern, based on the secondary structure and sequence features of the Cp GAIT element, was used to search a nonredundant 3′UTR database using the PatSearch program (bottom panel). Following the syntax of the PatSearch algorithm, allowed base-pairs are represented by r number and patterns defined by p number. The GAIT element-specific stems and loops are shown below. ( B ) PatSearch result predicted the presence of GAIT elements in Cp and VEGF-A 3′UTR. UTRdb ID refers to the sequence entry in the UTR database, and sequence position refers to the 3′UTR position of the sequence encoding the predicted GAIT element. ( C ) To show VEGF-A mRNA interaction with the GAIT complex in vivo , U937 cells were treated with IFN-γ for 8 or 24 h, and lysates were immunoprecipitated (IP) with anti-EPRS antibody to isolate GAIT complex, or with control pre-immune (Pre-im.) serum. RNA associated with the GAIT complex, or present in the non-immunoprecipitated supernatant (Sup.), was subjected to RT–PCR using primers specific for VEGF-A or β-actin mRNA, and products were resolved in 1.6% agarose gels. ( D ) To verify antibody specificity, lysate from U937 cells treated with IFN-γ for 24 h was immunoprecipitated with polyclonal anti-human EPRS antibody and immunoblotted with the same antibody, or with pre-immune serum as control.

    Techniques Used: Sequencing, In Vivo, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Silencing of VEGF-A translation in monocytic cells inhibits angiogenic activity. ( A ) EC proliferation was measured in presence of medium conditioned by IFN-γ-treated U937 cells. U937 cells were pre-treated with IFN-γ for up to 24 h, and then fresh medium was added for an additional 2 h. The conditioned medium was added to 50% confluent ECs, and proliferation measured by MTT assay. Cells were treated with recombinant VEGF-A (rVEGF-A, 10 ng/ml) as a positive control. Stimulation of proliferation was expressed as fold-increase compared to cells treated with medium alone (gray bars). Parallel wells contained conditioned medium pre-incubated with anti-VEGF-A antibody (black bars). Shown are the mean and standard deviation from three independent experiments. ( B ) Tube-formation by ECs on growth factor-depleted matrigel was determined after 12 h in presence of conditioned medium from U937 cells treated with IFN-γ for 8, 16, or 24 h, or with recombinant human VEGF-A (10 ng/ml). ( C ) EC tube formation was quantitated by computer-assisted tracing. Shown are the mean and standard deviation from three representative fields, for three independent experiments. ( D ) IFN-γ activates the transcription of VEGF-A, Cp, and other pro-inflammatory genes in macrophages at the site of chronic inflammation. Subsequently, IFN-γ activates the GAIT complex that binds to the GAIT element in the 3′UTR of VEGF-A, Cp, and possibly other transcripts, and silences their translation. This mechanism prevents persistent expression of these inflammatory proteins and reduces or resolves chronic inflammation and tissue injury.
    Figure Legend Snippet: Silencing of VEGF-A translation in monocytic cells inhibits angiogenic activity. ( A ) EC proliferation was measured in presence of medium conditioned by IFN-γ-treated U937 cells. U937 cells were pre-treated with IFN-γ for up to 24 h, and then fresh medium was added for an additional 2 h. The conditioned medium was added to 50% confluent ECs, and proliferation measured by MTT assay. Cells were treated with recombinant VEGF-A (rVEGF-A, 10 ng/ml) as a positive control. Stimulation of proliferation was expressed as fold-increase compared to cells treated with medium alone (gray bars). Parallel wells contained conditioned medium pre-incubated with anti-VEGF-A antibody (black bars). Shown are the mean and standard deviation from three independent experiments. ( B ) Tube-formation by ECs on growth factor-depleted matrigel was determined after 12 h in presence of conditioned medium from U937 cells treated with IFN-γ for 8, 16, or 24 h, or with recombinant human VEGF-A (10 ng/ml). ( C ) EC tube formation was quantitated by computer-assisted tracing. Shown are the mean and standard deviation from three representative fields, for three independent experiments. ( D ) IFN-γ activates the transcription of VEGF-A, Cp, and other pro-inflammatory genes in macrophages at the site of chronic inflammation. Subsequently, IFN-γ activates the GAIT complex that binds to the GAIT element in the 3′UTR of VEGF-A, Cp, and possibly other transcripts, and silences their translation. This mechanism prevents persistent expression of these inflammatory proteins and reduces or resolves chronic inflammation and tissue injury.

    Techniques Used: Activity Assay, MTT Assay, Recombinant, Positive Control, Incubation, Standard Deviation, Expressing

    6) Product Images from "Vascular adaptation to a dysfunctional endothelium as a consequence of Shb deficiency"

    Article Title: Vascular adaptation to a dysfunctional endothelium as a consequence of Shb deficiency

    Journal: Angiogenesis

    doi: 10.1007/s10456-012-9275-z

    Staining of wild-type and Shb knockout venules with VE-cadherin ( a, b ) and their ultrastructure visualized by scanning electron microscopy (SEM) ( c, d ). a Confocal microscopy of cremaster venules with or without treatment with VEGF-A after staining for
    Figure Legend Snippet: Staining of wild-type and Shb knockout venules with VE-cadherin ( a, b ) and their ultrastructure visualized by scanning electron microscopy (SEM) ( c, d ). a Confocal microscopy of cremaster venules with or without treatment with VEGF-A after staining for

    Techniques Used: Staining, Knock-Out, Electron Microscopy, Confocal Microscopy

    7) Product Images from "uPARAP/Endo180 receptor is a gatekeeper of VEGFR-2/VEGFR-3 heterodimerisation during pathological lymphangiogenesis"

    Article Title: uPARAP/Endo180 receptor is a gatekeeper of VEGFR-2/VEGFR-3 heterodimerisation during pathological lymphangiogenesis

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07514-1

    uPARAP deficiency stimulates VEGF-C-driven lymphangiogenesis. a Schematic of VEGF ligand interaction with VEGFR-2 and VEGFR-3 homodimers or VEGFR-2/VEGFR-3 heterodimers. b – e Gelatin sponges soaked with PBS ( b ), VEGF-C ( c ), VEGF-A ( d ), or mutated VEGF-C Cys156Ser ( e ) were implanted in mouse ears. White dots delineate the sponge in the ear. Lymphatic vasculature was examined by LYVE-1 (green) immunostaining. Histograms represent the area density of vessels quantified by a computer-assisted method and expressed as percentage of WT control ( b – d n = 6; e n = 8). Bars = 500 µm. f Indocyanin Green (ICG) clearance in VEGF-C-soaked sponges in uPARAP WT and KO mice using Xenogen IVIS. Histogram represents mean fluorescence signal detected at each time point ( n = 5). All results are expressed as mean ± SEM, and statistical analyses were performed using a non-parametric Mann–Whitney test. * P
    Figure Legend Snippet: uPARAP deficiency stimulates VEGF-C-driven lymphangiogenesis. a Schematic of VEGF ligand interaction with VEGFR-2 and VEGFR-3 homodimers or VEGFR-2/VEGFR-3 heterodimers. b – e Gelatin sponges soaked with PBS ( b ), VEGF-C ( c ), VEGF-A ( d ), or mutated VEGF-C Cys156Ser ( e ) were implanted in mouse ears. White dots delineate the sponge in the ear. Lymphatic vasculature was examined by LYVE-1 (green) immunostaining. Histograms represent the area density of vessels quantified by a computer-assisted method and expressed as percentage of WT control ( b – d n = 6; e n = 8). Bars = 500 µm. f Indocyanin Green (ICG) clearance in VEGF-C-soaked sponges in uPARAP WT and KO mice using Xenogen IVIS. Histogram represents mean fluorescence signal detected at each time point ( n = 5). All results are expressed as mean ± SEM, and statistical analyses were performed using a non-parametric Mann–Whitney test. * P

    Techniques Used: Immunostaining, Mouse Assay, Fluorescence, MANN-WHITNEY

    uPARAP downregulation impairs LEC chemotactic migration toward a VEGF-C gradient. LECs were transfected with a siRNA targeting uPARAP (siU1 and siU2) or a control siRNA (Ctr). RT-PCR ( a ) and Western blot ( b ) analyses of uPARAP, VEGFR-3 and VEGFR-2 expression (28S and GAPDH = internal controls). c Proliferation upon VEGF-C or VEGF-A stimulation. Data are those of one representative assay out of three ( n = 12 for T0 and n = 8 for T48 h, biological replicates). d Scratch assay in the presence of control medium ( n = 10), VEGF-C ( n = 10) or VEGF-A ( n = 3) (biological replicates in all conditions). e Phalloidin and uPARAP stainings of LECs migrating toward a VEGF-C or VEGF-A gradient in Ibidi µ-slides. Bars = 25 µm in left images and 50 µm in right images (higher magnification of inserts). f Boyden Chamber assay in the presence of control medium, wild type VEGF-C, VEGF-A, or mutated VEGF-C Cys156Ser . Histograms correspond to the mean ( n = 6 for control medium, VEGF-C and VEGF-A, and n = 3 for mutated VEGF-C Cys156Ser , biological replicates) ± SEM. g Chemotactic response to a gradient of complete medium, VEGF-C or VEGF-A. The rose diagram represents the direction of migration of more than 30 cells. Black arrows indicate the mean migration direction. The absence of arrow in VEGF-C diagrams reflects impaired directionality upon uPARAP silencing. Histograms represent the migratory speed (µm/min) where results are expressed as mean ( n = 6 biological replicates for complete medium and VEGF-C; n = 3 biological replicates for VEGF-A) ± SEM. Statistical analyses were performed using a non-parametric Mann–Whitney test. * P
    Figure Legend Snippet: uPARAP downregulation impairs LEC chemotactic migration toward a VEGF-C gradient. LECs were transfected with a siRNA targeting uPARAP (siU1 and siU2) or a control siRNA (Ctr). RT-PCR ( a ) and Western blot ( b ) analyses of uPARAP, VEGFR-3 and VEGFR-2 expression (28S and GAPDH = internal controls). c Proliferation upon VEGF-C or VEGF-A stimulation. Data are those of one representative assay out of three ( n = 12 for T0 and n = 8 for T48 h, biological replicates). d Scratch assay in the presence of control medium ( n = 10), VEGF-C ( n = 10) or VEGF-A ( n = 3) (biological replicates in all conditions). e Phalloidin and uPARAP stainings of LECs migrating toward a VEGF-C or VEGF-A gradient in Ibidi µ-slides. Bars = 25 µm in left images and 50 µm in right images (higher magnification of inserts). f Boyden Chamber assay in the presence of control medium, wild type VEGF-C, VEGF-A, or mutated VEGF-C Cys156Ser . Histograms correspond to the mean ( n = 6 for control medium, VEGF-C and VEGF-A, and n = 3 for mutated VEGF-C Cys156Ser , biological replicates) ± SEM. g Chemotactic response to a gradient of complete medium, VEGF-C or VEGF-A. The rose diagram represents the direction of migration of more than 30 cells. Black arrows indicate the mean migration direction. The absence of arrow in VEGF-C diagrams reflects impaired directionality upon uPARAP silencing. Histograms represent the migratory speed (µm/min) where results are expressed as mean ( n = 6 biological replicates for complete medium and VEGF-C; n = 3 biological replicates for VEGF-A) ± SEM. Statistical analyses were performed using a non-parametric Mann–Whitney test. * P

    Techniques Used: Migration, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Wound Healing Assay, Boyden Chamber Assay, MANN-WHITNEY

    8) Product Images from "An Angiotensin II Type 1 Receptor Blocker Prevents Renal Injury via Inhibition of the Notch Pathway in Ins2 Akita Diabetic Mice"

    Article Title: An Angiotensin II Type 1 Receptor Blocker Prevents Renal Injury via Inhibition of the Notch Pathway in Ins2 Akita Diabetic Mice

    Journal: Experimental Diabetes Research

    doi: 10.1155/2012/159874

    Telmisartan suppressed the activation of the Notch signaling pathway through inhibition of the angiotensin II type 1 receptor. The mRNA expression of Hes1, one of the Notch target genes; transforming growth factor β (TGF- β ); vascular endothelial growth factor-A (VEGF-A) were examined by reverse transcriptase-polymerase chain reaction. (a) The podocytes were stimulated with 10 −6 M Angiotensin II (AII) for 24 to 48 h. The mRNA expression of Hes1 increased in the presence of AII and peaked at 24 h. On the other hand, 10 −6 M telmisartan suppressed the AII-induced mRNA expression of Hes1 (upper panel). Quantification of the Hes1 mRNA expression compared to the internal control ( β -actin) (lower panel). (b) The podocytes were treated with 10 −6 M AII in the presence or absence of 10 −8 M candesartan for 24 h. Candesartan also suppressed the AII-induced mRNA expression of Hes1. (c) AII increased the TGF- β mRNA by 2.5-fold within 12 h. Telmisartan (10 −6 M) suppressed the expression of TGF- β significantly. (d) AII increased the VEGF-A expression by 2.0-fold. Telmisartan suppressed the expression of VEGF-A significantly. * P
    Figure Legend Snippet: Telmisartan suppressed the activation of the Notch signaling pathway through inhibition of the angiotensin II type 1 receptor. The mRNA expression of Hes1, one of the Notch target genes; transforming growth factor β (TGF- β ); vascular endothelial growth factor-A (VEGF-A) were examined by reverse transcriptase-polymerase chain reaction. (a) The podocytes were stimulated with 10 −6 M Angiotensin II (AII) for 24 to 48 h. The mRNA expression of Hes1 increased in the presence of AII and peaked at 24 h. On the other hand, 10 −6 M telmisartan suppressed the AII-induced mRNA expression of Hes1 (upper panel). Quantification of the Hes1 mRNA expression compared to the internal control ( β -actin) (lower panel). (b) The podocytes were treated with 10 −6 M AII in the presence or absence of 10 −8 M candesartan for 24 h. Candesartan also suppressed the AII-induced mRNA expression of Hes1. (c) AII increased the TGF- β mRNA by 2.5-fold within 12 h. Telmisartan (10 −6 M) suppressed the expression of TGF- β significantly. (d) AII increased the VEGF-A expression by 2.0-fold. Telmisartan suppressed the expression of VEGF-A significantly. * P

    Techniques Used: Activation Assay, Inhibition, Expressing, Polymerase Chain Reaction

    TGF- β and VEGF-A directly activated the Notch pathway. The podocytes were stimulated with 5 ng/mL transforming growth factor β (TGF- β ) or 10 ng/mL vascular endothelial growth factor-A (VEGF-A) in the presence or absence of 10 −6 M telmisartan. The mRNA expression of Hes1 was examined by reverse transcriptase-polymerase chain reaction. (a) TGF- β increased the expression of Hes1 irrespective of the presence or absence of telmisartan (upper panel). Quantification of Hes1 expression compared to the internal control ( β -actin). TGF- β significantly increased the Hes1 expression within 2 h by 2.1-fold (lower panel). (b) VEGF-A increased the expression of Hes1 irrespective of the presence or absence of telmisartan (upper panel). Quantification of the Hes1 expression compared to the internal control ( β -actin). VEGF-A significantly increased the Hes1 expression within 2 h by 1.6-fold (lower panel). * P
    Figure Legend Snippet: TGF- β and VEGF-A directly activated the Notch pathway. The podocytes were stimulated with 5 ng/mL transforming growth factor β (TGF- β ) or 10 ng/mL vascular endothelial growth factor-A (VEGF-A) in the presence or absence of 10 −6 M telmisartan. The mRNA expression of Hes1 was examined by reverse transcriptase-polymerase chain reaction. (a) TGF- β increased the expression of Hes1 irrespective of the presence or absence of telmisartan (upper panel). Quantification of Hes1 expression compared to the internal control ( β -actin). TGF- β significantly increased the Hes1 expression within 2 h by 2.1-fold (lower panel). (b) VEGF-A increased the expression of Hes1 irrespective of the presence or absence of telmisartan (upper panel). Quantification of the Hes1 expression compared to the internal control ( β -actin). VEGF-A significantly increased the Hes1 expression within 2 h by 1.6-fold (lower panel). * P

    Techniques Used: Expressing, Polymerase Chain Reaction

    9) Product Images from "Hypoxia and Extracellular Matrix Proteins Influence Angiogenesis and Lymphangiogenesis in Mouse Embryoid Bodies"

    Article Title: Hypoxia and Extracellular Matrix Proteins Influence Angiogenesis and Lymphangiogenesis in Mouse Embryoid Bodies

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2011.00103

    Double immunofluorescence staining of E22.5-day-old EBs in the presence of VEGF-A and VEGF-C . Representative images from three different experiments sets for each marker genes are shown. Arrowheads indicate MECA-32 positive blood vessel-like structures in E22.5-day-old EBs grown under both VN (A) and VH (B) treatments. Prox1 positive LECs are in close proximity to the blood vessel-like structures. Images were taken at 40× magnification (scale bar 30 μm).
    Figure Legend Snippet: Double immunofluorescence staining of E22.5-day-old EBs in the presence of VEGF-A and VEGF-C . Representative images from three different experiments sets for each marker genes are shown. Arrowheads indicate MECA-32 positive blood vessel-like structures in E22.5-day-old EBs grown under both VN (A) and VH (B) treatments. Prox1 positive LECs are in close proximity to the blood vessel-like structures. Images were taken at 40× magnification (scale bar 30 μm).

    Techniques Used: Double Immunofluorescence Staining, Marker

    E36.5-day-old EBs grown on laminin . EBs did not form vessel-like structures when stimulated under normoxic, hypoxic and VEGF-A/C environments. Representative data from three different experiments sets for each marker genes are shown. (A) Normoxic EBs grown on laminin did not show any distinguishable LEC phenotype, similar to those grown on collagen-I. (B) Hypoxic EBs did not induce any vessel-like structures within differentiating EBs. (C) N + VEGF-A/C EBs formed few ring-like structures (arrowheads) within the core of the EB and showed staining for LYVE1 in certain regions. (D) H + VEGF-A/C environments promoted LEC (LYVE1 + ) differentiation mostly on the periphery of differentiating EBs take. Images were en at 40× magnification (scale bar 30 μm).
    Figure Legend Snippet: E36.5-day-old EBs grown on laminin . EBs did not form vessel-like structures when stimulated under normoxic, hypoxic and VEGF-A/C environments. Representative data from three different experiments sets for each marker genes are shown. (A) Normoxic EBs grown on laminin did not show any distinguishable LEC phenotype, similar to those grown on collagen-I. (B) Hypoxic EBs did not induce any vessel-like structures within differentiating EBs. (C) N + VEGF-A/C EBs formed few ring-like structures (arrowheads) within the core of the EB and showed staining for LYVE1 in certain regions. (D) H + VEGF-A/C environments promoted LEC (LYVE1 + ) differentiation mostly on the periphery of differentiating EBs take. Images were en at 40× magnification (scale bar 30 μm).

    Techniques Used: Marker, Staining

    Double immunofluorescent staining of E36.5-day-old hypoxic EBs grown on collagen-I coverslips and treated with VEGF-A/C . Representative images from three different experiments sets for each marker genes are shown. (A,B) Collagen-I promotes predominantly the organization of LYVE1 positive lymphatic vessel-like structures (marked by arrowheads) when stimulated under hypoxic and VEGF-A/C environments. There are some traces of MECA-32 positive BECs along with these lymphatic vessel-like structures. Unlike E22.5-day-old EBs, all four types of treatment at E36.5 failed to form organized blood vessel-like structures. Images were taken at 40× magnification (scale bar 30 μm).
    Figure Legend Snippet: Double immunofluorescent staining of E36.5-day-old hypoxic EBs grown on collagen-I coverslips and treated with VEGF-A/C . Representative images from three different experiments sets for each marker genes are shown. (A,B) Collagen-I promotes predominantly the organization of LYVE1 positive lymphatic vessel-like structures (marked by arrowheads) when stimulated under hypoxic and VEGF-A/C environments. There are some traces of MECA-32 positive BECs along with these lymphatic vessel-like structures. Unlike E22.5-day-old EBs, all four types of treatment at E36.5 failed to form organized blood vessel-like structures. Images were taken at 40× magnification (scale bar 30 μm).

    Techniques Used: Staining, Marker

    Digital images (without magnification) of EBs at E22.5 grown under (A) N + VEGF-A/C (VN) and (B) H + VEGF-A/C (VH) treatments reveal contact of adjacent EBs to form cord-like structures, as marked by arrowheads . Representative result from 4 different experiment sets is shown.
    Figure Legend Snippet: Digital images (without magnification) of EBs at E22.5 grown under (A) N + VEGF-A/C (VN) and (B) H + VEGF-A/C (VH) treatments reveal contact of adjacent EBs to form cord-like structures, as marked by arrowheads . Representative result from 4 different experiment sets is shown.

    Techniques Used:

    Double immunostaining of E36.5-day-old EBs grown on collagen matrices subjected to three treatment types (N, H, VN) . Representative images from three different experiments sets for each marker genes are shown. (A) Normoxic EBs exhibited similar morphology to that observed at E22.5, illustrating differentiation of both BECs (MECA-32 + ) and LECs (Prox1 + ). (B) Hypoxia alone induces some lymphatic vessel-like formation, as marked by arrowheads. (C) Normoxic EBs treated with VEGF-A/C predominantly induces lymphatic vessel-like structures (arrowheads). Images were taken at 40× magnification (scale bar 30 μm).
    Figure Legend Snippet: Double immunostaining of E36.5-day-old EBs grown on collagen matrices subjected to three treatment types (N, H, VN) . Representative images from three different experiments sets for each marker genes are shown. (A) Normoxic EBs exhibited similar morphology to that observed at E22.5, illustrating differentiation of both BECs (MECA-32 + ) and LECs (Prox1 + ). (B) Hypoxia alone induces some lymphatic vessel-like formation, as marked by arrowheads. (C) Normoxic EBs treated with VEGF-A/C predominantly induces lymphatic vessel-like structures (arrowheads). Images were taken at 40× magnification (scale bar 30 μm).

    Techniques Used: Double Immunostaining, Marker

    10) Product Images from "Evidence of Th2 polarization of the sentinel lymph node (SLN) in melanoma"

    Article Title: Evidence of Th2 polarization of the sentinel lymph node (SLN) in melanoma

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2015.1026504

    Representative immunohistochemistry slides (5x) stained with anti-VEGF antibody from ( A ) benign nevi with no VEGF staining and ( B ) melanoma with diffuse cytoplasmic staining. ( C ) Histogram comparing VEGF receptor expression within melanoma SLN and control lymph nodes.
    Figure Legend Snippet: Representative immunohistochemistry slides (5x) stained with anti-VEGF antibody from ( A ) benign nevi with no VEGF staining and ( B ) melanoma with diffuse cytoplasmic staining. ( C ) Histogram comparing VEGF receptor expression within melanoma SLN and control lymph nodes.

    Techniques Used: Immunohistochemistry, Staining, Expressing

    Histogram demonstrating the functional enrichment z scores of Th1/Th2 pathways within CD4 + T cells of SLN draining melanoma compared to control LNs, respectively ( n = 4) on the left and CD4 + T cells of control lymph nodes treated with VEGF compared to untreated control lymph nodes on the right ( n = 3).
    Figure Legend Snippet: Histogram demonstrating the functional enrichment z scores of Th1/Th2 pathways within CD4 + T cells of SLN draining melanoma compared to control LNs, respectively ( n = 4) on the left and CD4 + T cells of control lymph nodes treated with VEGF compared to untreated control lymph nodes on the right ( n = 3).

    Techniques Used: Functional Assay

    11) Product Images from "Bone Marrow-Derived Mesenchymal Stem Cells Drive Lymphangiogenesis"

    Article Title: Bone Marrow-Derived Mesenchymal Stem Cells Drive Lymphangiogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106976

    VEGF-A is an important factor implicated in LEC stimulation by MSC conditioned medium. (A, B) The trapping of VEGF-A by the addition of soluble receptors-1 and -2 decreased MSC conditioned medium-induced LEC proliferation, measured by WST-1 assay (A) and migration in a Boyden chamber assay (B). *** P
    Figure Legend Snippet: VEGF-A is an important factor implicated in LEC stimulation by MSC conditioned medium. (A, B) The trapping of VEGF-A by the addition of soluble receptors-1 and -2 decreased MSC conditioned medium-induced LEC proliferation, measured by WST-1 assay (A) and migration in a Boyden chamber assay (B). *** P

    Techniques Used: WST-1 Assay, Migration, Boyden Chamber Assay

    VEGF-A secreted by BM-MSC activate LEC. (A) Western blot analyses of VEGF-A and VEGF-C production on serum-free EBM-2 (CTR) and MSC conditioned medium (MSC CM). (B) VEGFR-2 (top) and VEGFR-3 (bottom) proteins were detected following a phosphorylated tyrosine-containing protein (pY) immunoprecipation (IP) of LEC lysates after cell stimulation with control medium (CTR) or with MSC conditioned medium (MSC CM). Cells treated with VEGF-A (10 ng/ml) or VEGF-C (400 ng/ml) were used as negative and positive controls, respectively. GAPDH western blot was performed on the flowthrough of each sample.
    Figure Legend Snippet: VEGF-A secreted by BM-MSC activate LEC. (A) Western blot analyses of VEGF-A and VEGF-C production on serum-free EBM-2 (CTR) and MSC conditioned medium (MSC CM). (B) VEGFR-2 (top) and VEGFR-3 (bottom) proteins were detected following a phosphorylated tyrosine-containing protein (pY) immunoprecipation (IP) of LEC lysates after cell stimulation with control medium (CTR) or with MSC conditioned medium (MSC CM). Cells treated with VEGF-A (10 ng/ml) or VEGF-C (400 ng/ml) were used as negative and positive controls, respectively. GAPDH western blot was performed on the flowthrough of each sample.

    Techniques Used: Western Blot, Cell Stimulation

    12) Product Images from "Semaphorin 6A regulates angiogenesis by modulating VEGF signaling"

    Article Title: Semaphorin 6A regulates angiogenesis by modulating VEGF signaling

    Journal: Blood

    doi: 10.1182/blood-2012-02-410076

    VEGF-A and FGF2 signaling in Sema6A -silenced endothelial cells. (A) Control and Sema6A -silenced HUVECs were incubated 15 minutes with VEGF-A (100 ng/mL). Cell lysates were tested for phosphorylated and total VEGFR2, AKT, and ERK1/2; β-actin staining
    Figure Legend Snippet: VEGF-A and FGF2 signaling in Sema6A -silenced endothelial cells. (A) Control and Sema6A -silenced HUVECs were incubated 15 minutes with VEGF-A (100 ng/mL). Cell lysates were tested for phosphorylated and total VEGFR2, AKT, and ERK1/2; β-actin staining

    Techniques Used: Incubation, Staining

    Endothelial cells require VEGF signaling for survival. (A) VEGFA gene expression in Sema6A -silenced and control HUVECs was measured by quantitative PCR. The results reflect the mean (± SEM; n = 4) relative mRNA levels. (B) HUVECs were incubated
    Figure Legend Snippet: Endothelial cells require VEGF signaling for survival. (A) VEGFA gene expression in Sema6A -silenced and control HUVECs was measured by quantitative PCR. The results reflect the mean (± SEM; n = 4) relative mRNA levels. (B) HUVECs were incubated

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Incubation

    13) Product Images from "A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity"

    Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7601774

    The 3′UTR of VEGF-A mRNA mediates translation inhibition. ( A ) Schematic of VEGF-A mRNA and chimeric luciferase constructs used for in vitro translation (top panel). The m 7 G cap is indicated by an open circle, the IRES by a light gray rectangle, the putative GAIT element by a black rectangle, and the AREs by dark gray rectangles. Capped, FLuc-VEGF-A 3′UTR (11–900) -A 30 RNA was translated in RRL containing [ 35 S]Met, and in absence or presence of cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h (middle panel). Capped, RLuc RNA lacking the GAIT element was co-translated in each reaction as control. Translation reactions were resolved on SDS–10% polyacrylamide gel. The same RNAs were translated in the presence of cytosolic extract from 24-h, IFN-γ-treated U937 cells, and in the presence of 10- and 50-fold molar excess of in vitro transcribed VEGF-A 3′UTR RNA as competitor (bottom panel). ( B ) Schematic of chimeric luciferase constructs used for in vitro translation (top panel). In vitro translation, in presence of IFN-γ-treated U937 cytosolic extracts, of capped FLuc-VEGF-A 3′UTR (324–455) -A 30 encompassing the putative GAIT element (middle panel), and FLuc-VEGF-A 3′UTR (441–560) -A 30 (bottom panel). RLuc RNA was co-translated in each reaction.
    Figure Legend Snippet: The 3′UTR of VEGF-A mRNA mediates translation inhibition. ( A ) Schematic of VEGF-A mRNA and chimeric luciferase constructs used for in vitro translation (top panel). The m 7 G cap is indicated by an open circle, the IRES by a light gray rectangle, the putative GAIT element by a black rectangle, and the AREs by dark gray rectangles. Capped, FLuc-VEGF-A 3′UTR (11–900) -A 30 RNA was translated in RRL containing [ 35 S]Met, and in absence or presence of cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h (middle panel). Capped, RLuc RNA lacking the GAIT element was co-translated in each reaction as control. Translation reactions were resolved on SDS–10% polyacrylamide gel. The same RNAs were translated in the presence of cytosolic extract from 24-h, IFN-γ-treated U937 cells, and in the presence of 10- and 50-fold molar excess of in vitro transcribed VEGF-A 3′UTR RNA as competitor (bottom panel). ( B ) Schematic of chimeric luciferase constructs used for in vitro translation (top panel). In vitro translation, in presence of IFN-γ-treated U937 cytosolic extracts, of capped FLuc-VEGF-A 3′UTR (324–455) -A 30 encompassing the putative GAIT element (middle panel), and FLuc-VEGF-A 3′UTR (441–560) -A 30 (bottom panel). RLuc RNA was co-translated in each reaction.

    Techniques Used: Inhibition, Luciferase, Construct, In Vitro

    Translational silencing of VEGF-A expression in vivo. ( A ) RT–PCR analysis of total RNA from U937 cells treated with IFN-γ for 0, 8, or 24 h. RT–PCR was done using primers specific for VEGF-A (top panel) and GAPDH (bottom panel). Real-time PCR results indicating the increase in VEGF-A mRNA expression in IFN-γ-treated cells compared to untreated cells are included below the top panel (expressed as fold-increase normalized to β-actin). ( B ) Cell lysates from U937 cells treated with IFN-γ for 0, 8, or 24 h were processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top) and anti-GAPDH (bottom panel) antibodies. ( C ) RT–PCR analysis of total RNA from human PBMCs treated with IFN-γ for 0, 8, or 24 h. RT–PCR was performed using primers specific for VEGF-A (top panel) and GAPDH (bottom panel). ( D ) Cell lysates from PBMCs treated with IFN-γ for 0, 8, or 24 h were processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top) and anti-GAPDH (bottom panel) antibodies. ( E ) U937 cells were treated with IFN-γ for up to 24 h. At the end of each interval, cells were metabolically labeled with [ 35 S]Met/Cys for 1 h. Conditioned media and cell lysates were immunoprecipitated with anti-VEGF-A antibody and resolved by electrophoresis on SDS–10% polyacrylamide gel (top panel). Monomeric and dimeric VEGF-A forms are indicated by arrows. The same samples were subjected to electrophoresis without immunoprecipitation (bottom). ( F ) U937 cells were treated with IFN-γ for 8 or 24 h and cytosolic extracts were fractionated into polysomal and non-polysomal, RNP fractions by ultracentrifugation on a 20% sucrose cushion in the presence or absence of 10 mM EDTA. RNA associated with each fraction was isolated and subjected to RT–PCR using primers specific for VEGF-A (top panel) and GAPDH (bottom panel).
    Figure Legend Snippet: Translational silencing of VEGF-A expression in vivo. ( A ) RT–PCR analysis of total RNA from U937 cells treated with IFN-γ for 0, 8, or 24 h. RT–PCR was done using primers specific for VEGF-A (top panel) and GAPDH (bottom panel). Real-time PCR results indicating the increase in VEGF-A mRNA expression in IFN-γ-treated cells compared to untreated cells are included below the top panel (expressed as fold-increase normalized to β-actin). ( B ) Cell lysates from U937 cells treated with IFN-γ for 0, 8, or 24 h were processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top) and anti-GAPDH (bottom panel) antibodies. ( C ) RT–PCR analysis of total RNA from human PBMCs treated with IFN-γ for 0, 8, or 24 h. RT–PCR was performed using primers specific for VEGF-A (top panel) and GAPDH (bottom panel). ( D ) Cell lysates from PBMCs treated with IFN-γ for 0, 8, or 24 h were processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top) and anti-GAPDH (bottom panel) antibodies. ( E ) U937 cells were treated with IFN-γ for up to 24 h. At the end of each interval, cells were metabolically labeled with [ 35 S]Met/Cys for 1 h. Conditioned media and cell lysates were immunoprecipitated with anti-VEGF-A antibody and resolved by electrophoresis on SDS–10% polyacrylamide gel (top panel). Monomeric and dimeric VEGF-A forms are indicated by arrows. The same samples were subjected to electrophoresis without immunoprecipitation (bottom). ( F ) U937 cells were treated with IFN-γ for 8 or 24 h and cytosolic extracts were fractionated into polysomal and non-polysomal, RNP fractions by ultracentrifugation on a 20% sucrose cushion in the presence or absence of 10 mM EDTA. RNA associated with each fraction was isolated and subjected to RT–PCR using primers specific for VEGF-A (top panel) and GAPDH (bottom panel).

    Techniques Used: Expressing, In Vivo, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Metabolic Labelling, Labeling, Immunoprecipitation, Electrophoresis, Isolation

    The GAIT complex binds the VEGF-A GAIT element and causes translational silencing. ( A ) RNA EMSA using 32 P-labeled Cp and VEGF-A GAIT element probes. The riboprobes were incubated with cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h. RNA–protein complexes were resolved by electrophoresis on a nondenaturing 5% polyacrylamide gel. ( B ) RNA–protein complexes formed between 32 P-labeled VEGF-A GAIT element RNA and lysates from 24-h, IFN-γ-treated U937 cells were supershifted with antibodies against GAIT complex components. The cell lysate was incubated with the respective antibodies or non-immune IgG before incubation with the riboprobe. ( C ) Lysate from U937 cells treated with IFN-γ for 24 h was incubated with protein-A Sepharose beads coupled to anti-EPRS antibody (or to pre-immune serum, Pre-im.) to immunodeplete the GAIT complex. The beads were pelleted, and the supernatant subjected to immunoblotting with anti-EPRS antibody to verify effective immunodepletion. ( D ) At 24-h, IFN-γ-treated U937 cell lysates, immunodepleted with anti-EPRS antibody or pre-immune serum, were added to in vitro translation reactions containing FLuc-VEGF-A 3′UTR (11–900) -A 30 and RLuc RNAs.
    Figure Legend Snippet: The GAIT complex binds the VEGF-A GAIT element and causes translational silencing. ( A ) RNA EMSA using 32 P-labeled Cp and VEGF-A GAIT element probes. The riboprobes were incubated with cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h. RNA–protein complexes were resolved by electrophoresis on a nondenaturing 5% polyacrylamide gel. ( B ) RNA–protein complexes formed between 32 P-labeled VEGF-A GAIT element RNA and lysates from 24-h, IFN-γ-treated U937 cells were supershifted with antibodies against GAIT complex components. The cell lysate was incubated with the respective antibodies or non-immune IgG before incubation with the riboprobe. ( C ) Lysate from U937 cells treated with IFN-γ for 24 h was incubated with protein-A Sepharose beads coupled to anti-EPRS antibody (or to pre-immune serum, Pre-im.) to immunodeplete the GAIT complex. The beads were pelleted, and the supernatant subjected to immunoblotting with anti-EPRS antibody to verify effective immunodepletion. ( D ) At 24-h, IFN-γ-treated U937 cell lysates, immunodepleted with anti-EPRS antibody or pre-immune serum, were added to in vitro translation reactions containing FLuc-VEGF-A 3′UTR (11–900) -A 30 and RLuc RNAs.

    Techniques Used: Labeling, Incubation, Electrophoresis, In Vitro

    Functional identification of the VEGF-A 3′UTR GAIT element. ( A ) Folding structures of the Cp (nt 78–106) and the putative VEGF-A GAIT (nt 358–386) elements as predicted by the Mfold algorithm. Base pairing between A7:U23 and U8:A22 was disallowed while folding the VEGF-A GAIT element. ( B ) Chimeric luciferase constructs containing wild-type or mutant VEGF-A 3′UTR GAIT elements. Capped and poly-A tailed RNAs, containing the putative VEGF-A GAIT element (FLuc-VEGF-A GAIT-A 30 ) or a mutant (U10C) GAIT element (FLuc-VEGF-A GAIT mut -A 30 ), downstream of FLuc (top panel), were subjected to in vitro translation in presence of cytosolic extracts from IFN-γ-treated U937 cells (bottom panel). RLuc RNA was co-translated in each reaction. ( C ) Capped and poly-A tailed RNAs, containing the putative VEGF-A GAIT element or a mutant GAIT element as in (B), were subjected to in vitro translation in presence of cytosolic extracts from IFN-γ-treated human PBMC (top panel). RLuc RNA was co-translated in each reaction. Fluc was quantified by densitometry, normalized to Rluc, and expressed as per cent of control condition without cell lysate (bottom). ( D ) U937 cells were transfected with eukaryotic, CMV-driven expression vectors containing the FLuc gene upstream of either wild-type (CMV-FLuc-VEGF-A GAIT-A 30 ) or mutant VEGF-A GAIT element (CMV-FLuc-VEGF-A GAIT mut -A 30 ) or lacking any GAIT element (CMV-FLuc). Cells were co-transfected with a vector containing RLuc gene under the SV40 promoter. Following transfection, cells were treated with IFN-γ for 8 (gray bars) or 24 h (black bars), or with medium alone (hatched bars). Luciferase activity in cell lysates was measured by dual luciferase assay. Results show mean and standard deviation of values from three independent experiments.
    Figure Legend Snippet: Functional identification of the VEGF-A 3′UTR GAIT element. ( A ) Folding structures of the Cp (nt 78–106) and the putative VEGF-A GAIT (nt 358–386) elements as predicted by the Mfold algorithm. Base pairing between A7:U23 and U8:A22 was disallowed while folding the VEGF-A GAIT element. ( B ) Chimeric luciferase constructs containing wild-type or mutant VEGF-A 3′UTR GAIT elements. Capped and poly-A tailed RNAs, containing the putative VEGF-A GAIT element (FLuc-VEGF-A GAIT-A 30 ) or a mutant (U10C) GAIT element (FLuc-VEGF-A GAIT mut -A 30 ), downstream of FLuc (top panel), were subjected to in vitro translation in presence of cytosolic extracts from IFN-γ-treated U937 cells (bottom panel). RLuc RNA was co-translated in each reaction. ( C ) Capped and poly-A tailed RNAs, containing the putative VEGF-A GAIT element or a mutant GAIT element as in (B), were subjected to in vitro translation in presence of cytosolic extracts from IFN-γ-treated human PBMC (top panel). RLuc RNA was co-translated in each reaction. Fluc was quantified by densitometry, normalized to Rluc, and expressed as per cent of control condition without cell lysate (bottom). ( D ) U937 cells were transfected with eukaryotic, CMV-driven expression vectors containing the FLuc gene upstream of either wild-type (CMV-FLuc-VEGF-A GAIT-A 30 ) or mutant VEGF-A GAIT element (CMV-FLuc-VEGF-A GAIT mut -A 30 ) or lacking any GAIT element (CMV-FLuc). Cells were co-transfected with a vector containing RLuc gene under the SV40 promoter. Following transfection, cells were treated with IFN-γ for 8 (gray bars) or 24 h (black bars), or with medium alone (hatched bars). Luciferase activity in cell lysates was measured by dual luciferase assay. Results show mean and standard deviation of values from three independent experiments.

    Techniques Used: Functional Assay, Luciferase, Construct, Mutagenesis, In Vitro, Transfection, Expressing, Plasmid Preparation, Activity Assay, Standard Deviation

    Ablation of the GAIT complex in vivo prevents translational silencing of VEGF-A. ( A ) Lysates from U937 cells stably transfected with pSUPER vector (U937-pSUPER) or pSUPER encoding a short hairpin RNA targeting L13a (U937-L13a-SHR) were immunoblotted with anti-L13a antibody. ( B ) Lysates from the stably transfected cell lines in (A) were treated with IFN-γ for 0, 8, or 24 h and processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top panel) and anti-GAPDH (bottom panel) antibodies. ( C ) Total RNA was isolated from the stably transfected cell lines treated with IFN-γ for 0, 8, or 24 h, and analyzed by RT–PCR using primers specific for VEGF-A (top panel) and β-actin (bottom panel). Real-time PCR results indicating increased VEGF-A mRNA expression in IFN-γ-treated cells compared to untreated cells (expressed as fold-increase normalized to β-actin) are inserted below the top panel. ( D ) The cell lines described in (A) were treated with IFN-γ for 24 h and lysates immunoprecipitated with anti-EPRS antibody, followed by RT–PCR with VEGF-A-specific primers.
    Figure Legend Snippet: Ablation of the GAIT complex in vivo prevents translational silencing of VEGF-A. ( A ) Lysates from U937 cells stably transfected with pSUPER vector (U937-pSUPER) or pSUPER encoding a short hairpin RNA targeting L13a (U937-L13a-SHR) were immunoblotted with anti-L13a antibody. ( B ) Lysates from the stably transfected cell lines in (A) were treated with IFN-γ for 0, 8, or 24 h and processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top panel) and anti-GAPDH (bottom panel) antibodies. ( C ) Total RNA was isolated from the stably transfected cell lines treated with IFN-γ for 0, 8, or 24 h, and analyzed by RT–PCR using primers specific for VEGF-A (top panel) and β-actin (bottom panel). Real-time PCR results indicating increased VEGF-A mRNA expression in IFN-γ-treated cells compared to untreated cells (expressed as fold-increase normalized to β-actin) are inserted below the top panel. ( D ) The cell lines described in (A) were treated with IFN-γ for 24 h and lysates immunoprecipitated with anti-EPRS antibody, followed by RT–PCR with VEGF-A-specific primers.

    Techniques Used: In Vivo, Stable Transfection, Transfection, Plasmid Preparation, shRNA, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, Immunoprecipitation

    VEGF-A mRNA interacts with the GAIT complex. ( A ) Secondary structure and sequence features of the human Cp GAIT element (top panel). The query pattern, based on the secondary structure and sequence features of the Cp GAIT element, was used to search a nonredundant 3′UTR database using the PatSearch program (bottom panel). Following the syntax of the PatSearch algorithm, allowed base-pairs are represented by r number and patterns defined by p number. The GAIT element-specific stems and loops are shown below. ( B ) PatSearch result predicted the presence of GAIT elements in Cp and VEGF-A 3′UTR. UTRdb ID refers to the sequence entry in the UTR database, and sequence position refers to the 3′UTR position of the sequence encoding the predicted GAIT element. ( C ) To show VEGF-A mRNA interaction with the GAIT complex in vivo , U937 cells were treated with IFN-γ for 8 or 24 h, and lysates were immunoprecipitated (IP) with anti-EPRS antibody to isolate GAIT complex, or with control pre-immune (Pre-im.) serum. RNA associated with the GAIT complex, or present in the non-immunoprecipitated supernatant (Sup.), was subjected to RT–PCR using primers specific for VEGF-A or β-actin mRNA, and products were resolved in 1.6% agarose gels. ( D ) To verify antibody specificity, lysate from U937 cells treated with IFN-γ for 24 h was immunoprecipitated with polyclonal anti-human EPRS antibody and immunoblotted with the same antibody, or with pre-immune serum as control.
    Figure Legend Snippet: VEGF-A mRNA interacts with the GAIT complex. ( A ) Secondary structure and sequence features of the human Cp GAIT element (top panel). The query pattern, based on the secondary structure and sequence features of the Cp GAIT element, was used to search a nonredundant 3′UTR database using the PatSearch program (bottom panel). Following the syntax of the PatSearch algorithm, allowed base-pairs are represented by r number and patterns defined by p number. The GAIT element-specific stems and loops are shown below. ( B ) PatSearch result predicted the presence of GAIT elements in Cp and VEGF-A 3′UTR. UTRdb ID refers to the sequence entry in the UTR database, and sequence position refers to the 3′UTR position of the sequence encoding the predicted GAIT element. ( C ) To show VEGF-A mRNA interaction with the GAIT complex in vivo , U937 cells were treated with IFN-γ for 8 or 24 h, and lysates were immunoprecipitated (IP) with anti-EPRS antibody to isolate GAIT complex, or with control pre-immune (Pre-im.) serum. RNA associated with the GAIT complex, or present in the non-immunoprecipitated supernatant (Sup.), was subjected to RT–PCR using primers specific for VEGF-A or β-actin mRNA, and products were resolved in 1.6% agarose gels. ( D ) To verify antibody specificity, lysate from U937 cells treated with IFN-γ for 24 h was immunoprecipitated with polyclonal anti-human EPRS antibody and immunoblotted with the same antibody, or with pre-immune serum as control.

    Techniques Used: Sequencing, In Vivo, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Silencing of VEGF-A translation in monocytic cells inhibits angiogenic activity. ( A ) EC proliferation was measured in presence of medium conditioned by IFN-γ-treated U937 cells. U937 cells were pre-treated with IFN-γ for up to 24 h, and then fresh medium was added for an additional 2 h. The conditioned medium was added to 50% confluent ECs, and proliferation measured by MTT assay. Cells were treated with recombinant VEGF-A (rVEGF-A, 10 ng/ml) as a positive control. Stimulation of proliferation was expressed as fold-increase compared to cells treated with medium alone (gray bars). Parallel wells contained conditioned medium pre-incubated with anti-VEGF-A antibody (black bars). Shown are the mean and standard deviation from three independent experiments. ( B ) Tube-formation by ECs on growth factor-depleted matrigel was determined after 12 h in presence of conditioned medium from U937 cells treated with IFN-γ for 8, 16, or 24 h, or with recombinant human VEGF-A (10 ng/ml). ( C ) EC tube formation was quantitated by computer-assisted tracing. Shown are the mean and standard deviation from three representative fields, for three independent experiments. ( D ) IFN-γ activates the transcription of VEGF-A, Cp, and other pro-inflammatory genes in macrophages at the site of chronic inflammation. Subsequently, IFN-γ activates the GAIT complex that binds to the GAIT element in the 3′UTR of VEGF-A, Cp, and possibly other transcripts, and silences their translation. This mechanism prevents persistent expression of these inflammatory proteins and reduces or resolves chronic inflammation and tissue injury.
    Figure Legend Snippet: Silencing of VEGF-A translation in monocytic cells inhibits angiogenic activity. ( A ) EC proliferation was measured in presence of medium conditioned by IFN-γ-treated U937 cells. U937 cells were pre-treated with IFN-γ for up to 24 h, and then fresh medium was added for an additional 2 h. The conditioned medium was added to 50% confluent ECs, and proliferation measured by MTT assay. Cells were treated with recombinant VEGF-A (rVEGF-A, 10 ng/ml) as a positive control. Stimulation of proliferation was expressed as fold-increase compared to cells treated with medium alone (gray bars). Parallel wells contained conditioned medium pre-incubated with anti-VEGF-A antibody (black bars). Shown are the mean and standard deviation from three independent experiments. ( B ) Tube-formation by ECs on growth factor-depleted matrigel was determined after 12 h in presence of conditioned medium from U937 cells treated with IFN-γ for 8, 16, or 24 h, or with recombinant human VEGF-A (10 ng/ml). ( C ) EC tube formation was quantitated by computer-assisted tracing. Shown are the mean and standard deviation from three representative fields, for three independent experiments. ( D ) IFN-γ activates the transcription of VEGF-A, Cp, and other pro-inflammatory genes in macrophages at the site of chronic inflammation. Subsequently, IFN-γ activates the GAIT complex that binds to the GAIT element in the 3′UTR of VEGF-A, Cp, and possibly other transcripts, and silences their translation. This mechanism prevents persistent expression of these inflammatory proteins and reduces or resolves chronic inflammation and tissue injury.

    Techniques Used: Activity Assay, MTT Assay, Recombinant, Positive Control, Incubation, Standard Deviation, Expressing

    14) Product Images from "IL-25 induces airways angiogenesis and expression of multiple angiogenic factors in a murine asthma model"

    Article Title: IL-25 induces airways angiogenesis and expression of multiple angiogenic factors in a murine asthma model

    Journal: Respiratory Research

    doi: 10.1186/s12931-015-0197-3

    IL-25, but not IL-4, IL-5 or IL-13 induced angiogenesis in vitro. Top panel: representative light photomicrographs (4x original magnification) show formation of primitive vascular tubule structures by human vascular endothelial cells after 11 days of culture (top panel) with medium (A) , VEGF (10 ng/mL) (B) , IL-25 (10 ng/mL) (C) , IL-4 (10 ng/mL) (D) , IL-5 (10 ng/mL) (E) and IL-13 (10 ng/mL) (F) . Bottom panel: computer-assisted quantification of total tubule lengths (G) , numbers of branch points (H) and total numbers of tubules (I) . Bars show the mean ± SEM of three separate experiments performed in duplicate. *p
    Figure Legend Snippet: IL-25, but not IL-4, IL-5 or IL-13 induced angiogenesis in vitro. Top panel: representative light photomicrographs (4x original magnification) show formation of primitive vascular tubule structures by human vascular endothelial cells after 11 days of culture (top panel) with medium (A) , VEGF (10 ng/mL) (B) , IL-25 (10 ng/mL) (C) , IL-4 (10 ng/mL) (D) , IL-5 (10 ng/mL) (E) and IL-13 (10 ng/mL) (F) . Bottom panel: computer-assisted quantification of total tubule lengths (G) , numbers of branch points (H) and total numbers of tubules (I) . Bars show the mean ± SEM of three separate experiments performed in duplicate. *p

    Techniques Used: In Vitro

    IL-25 potentiated airways VEGF and bFGF expression. Concentrations of basic fibroblast growth factor (bFGF, A and B ) and vascular endothelial growth factor (VEGF, C and D ) in bronchoalveolar lavage fluid (BALF) and lung tissue homogenates from saline (NS)-, OVA- and IL-25-challenged mice at various time points as indicated. The data were collected from 3 independent experiments and are expressed as the mean ± SEM (n = 5 in each group at each time point). *p
    Figure Legend Snippet: IL-25 potentiated airways VEGF and bFGF expression. Concentrations of basic fibroblast growth factor (bFGF, A and B ) and vascular endothelial growth factor (VEGF, C and D ) in bronchoalveolar lavage fluid (BALF) and lung tissue homogenates from saline (NS)-, OVA- and IL-25-challenged mice at various time points as indicated. The data were collected from 3 independent experiments and are expressed as the mean ± SEM (n = 5 in each group at each time point). *p

    Techniques Used: Expressing, Mouse Assay

    15) Product Images from "Angiopoietin-4 Inhibits Angiogenesis and Reduces Interstitial Fluid Pressure 1"

    Article Title: Angiopoietin-4 Inhibits Angiogenesis and Reduces Interstitial Fluid Pressure 1

    Journal: Neoplasia (New York, N.Y.)

    doi:

    RT-CES measurements of HUVEC proliferation are shown. The combination of bFGF (10 ng/ml) and VEGF (3 ng/ml) significantly induced HUVEC proliferation (A and B). There was no significant effect of Ang-4 addition, neither when Ang-4 was applied in combination with bFGF and VEGF (A) nor when Ang-4 was applied as a single agent (B). After 24 hours, proliferation curves level off, and cell growth is no longer exponential. Beyond 24 hours, the bottom of the wells is completely covered by ECs, and there may be lack of media and growth factors. As expected, the lowest proliferation rate was found in the wells containing FBS alone, with no growth factors added.
    Figure Legend Snippet: RT-CES measurements of HUVEC proliferation are shown. The combination of bFGF (10 ng/ml) and VEGF (3 ng/ml) significantly induced HUVEC proliferation (A and B). There was no significant effect of Ang-4 addition, neither when Ang-4 was applied in combination with bFGF and VEGF (A) nor when Ang-4 was applied as a single agent (B). After 24 hours, proliferation curves level off, and cell growth is no longer exponential. Beyond 24 hours, the bottom of the wells is completely covered by ECs, and there may be lack of media and growth factors. As expected, the lowest proliferation rate was found in the wells containing FBS alone, with no growth factors added.

    Techniques Used:

    In vivo angiogenic response in Matrigel chambers. (A) Growth factor-induced angiogenesis in Matrigel chambers. The combination of 750 ng/ml bFGF and 250 ng/ml VEGF induced angiogenesis on day 12 significantly above the control level. Addition of Ang-4 (500 ng/ml) significantly inhibited the response. (B) Tumor angiogenesis in Matrigel chambers induced by GLC19 SCLC cells. Tumor cells induced a highly significant angiogenic response by 16 days of implantation in Matrigel chambers. The addition of Ang-4 (1500 ng/ml) to tumor cells significantly inhibited angiogenic response. (C) Angiogenesis in Matrigel chambers induced by transfected tumor cells. Ang-4-transfected GLC19 cells induced an angiogenic response that was significantly lower than that of mock-transfected cells. Bevacizumab treatment of the mock-transfected cells also reduced angiogenic response. Each column represents a mean value of 18 to 20 chambers in each group. Data are expressed as mean ± SD.
    Figure Legend Snippet: In vivo angiogenic response in Matrigel chambers. (A) Growth factor-induced angiogenesis in Matrigel chambers. The combination of 750 ng/ml bFGF and 250 ng/ml VEGF induced angiogenesis on day 12 significantly above the control level. Addition of Ang-4 (500 ng/ml) significantly inhibited the response. (B) Tumor angiogenesis in Matrigel chambers induced by GLC19 SCLC cells. Tumor cells induced a highly significant angiogenic response by 16 days of implantation in Matrigel chambers. The addition of Ang-4 (1500 ng/ml) to tumor cells significantly inhibited angiogenic response. (C) Angiogenesis in Matrigel chambers induced by transfected tumor cells. Ang-4-transfected GLC19 cells induced an angiogenic response that was significantly lower than that of mock-transfected cells. Bevacizumab treatment of the mock-transfected cells also reduced angiogenic response. Each column represents a mean value of 18 to 20 chambers in each group. Data are expressed as mean ± SD.

    Techniques Used: In Vivo, Transfection

    In vitro EC migration in Boyden chambers. (A) Addition of 40 ng/ml Ang-4 to the serum-free medium significantly reduced the number of migrated cells from 76.2 to 41.5. Further addition of Ang-4 did not result in further inhibition of EC migration. (B) In a separate experiment, the combination of 50 ng/ml bFGF and 16 ng/ml VEGF caused EC migration significantly above the control level (mean, 96.1 vs 50.2 cells/field). This response was significantly inhibited by the addition of Ang-4 (40 ng/ml). Data are expressed as mean ± SD. Each column represents a mean value of six wells in each group. Migrated cells were counted at x250 magnification.
    Figure Legend Snippet: In vitro EC migration in Boyden chambers. (A) Addition of 40 ng/ml Ang-4 to the serum-free medium significantly reduced the number of migrated cells from 76.2 to 41.5. Further addition of Ang-4 did not result in further inhibition of EC migration. (B) In a separate experiment, the combination of 50 ng/ml bFGF and 16 ng/ml VEGF caused EC migration significantly above the control level (mean, 96.1 vs 50.2 cells/field). This response was significantly inhibited by the addition of Ang-4 (40 ng/ml). Data are expressed as mean ± SD. Each column represents a mean value of six wells in each group. Migrated cells were counted at x250 magnification.

    Techniques Used: In Vitro, Migration, Inhibition

    16) Product Images from "IGF2 and IGF1R identified as novel tip cell genes in primary microvascular endothelial cell monolayers"

    Article Title: IGF2 and IGF1R identified as novel tip cell genes in primary microvascular endothelial cell monolayers

    Journal: Angiogenesis

    doi: 10.1007/s10456-018-9627-4

    IGF2 and IGF1R are essential for CD34 + tip cell fate. a, b Effect of knockdown of IGF2 and IGF1R expression on percentages of CD34 + tip cells. Bars show percentages of CD34 + hMVECs ( a ) and HUVECs ( b ) treated with siNT, siIGF2 , or siIGF1R as detected by flow cytometry. c, d Quantification of numbers of sprouts ( c ) and average sprout length ( d ) of spheroids composed of HUVECs after treatment with siNT, siIGF2 , or siIGF1R . e Analysis of CD34 + tip cell morphology after knockdown of IGF2 and IGF1R expression. Staining of CD34 (green), F-actin (phalloidin, red), and nuclei (DAPI, blue) in hMVECs. Scale bars represent 50 µm (first 3 columns) and 100 µm (last column). f Effect of rhIGF2 on CD34 + HUVEC tip cell percentages. Bars shows CD34 + tip cells of HUVECs treated with either 25 ng/mL BSA, 25 ng/mL VEGF-A, or 50 ng/mL rhIGF2 as detected by flow cytometry. g, h Effects of VEGF and IGF2 on the number of sprouts ( g ) and average sprout length ( h ) in spheroids of CD34 + tip cells or CD34 − non-tip cells. Data in a – d and f – h are shown as mean ± standard deviation after factor correction. * p
    Figure Legend Snippet: IGF2 and IGF1R are essential for CD34 + tip cell fate. a, b Effect of knockdown of IGF2 and IGF1R expression on percentages of CD34 + tip cells. Bars show percentages of CD34 + hMVECs ( a ) and HUVECs ( b ) treated with siNT, siIGF2 , or siIGF1R as detected by flow cytometry. c, d Quantification of numbers of sprouts ( c ) and average sprout length ( d ) of spheroids composed of HUVECs after treatment with siNT, siIGF2 , or siIGF1R . e Analysis of CD34 + tip cell morphology after knockdown of IGF2 and IGF1R expression. Staining of CD34 (green), F-actin (phalloidin, red), and nuclei (DAPI, blue) in hMVECs. Scale bars represent 50 µm (first 3 columns) and 100 µm (last column). f Effect of rhIGF2 on CD34 + HUVEC tip cell percentages. Bars shows CD34 + tip cells of HUVECs treated with either 25 ng/mL BSA, 25 ng/mL VEGF-A, or 50 ng/mL rhIGF2 as detected by flow cytometry. g, h Effects of VEGF and IGF2 on the number of sprouts ( g ) and average sprout length ( h ) in spheroids of CD34 + tip cells or CD34 − non-tip cells. Data in a – d and f – h are shown as mean ± standard deviation after factor correction. * p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Standard Deviation

    Human microvascular endothelial cell cultures contain CD34 + tip cells. a Identification of tip cells by staining with anti-CD34 (green), F-actin (phalloidin, red), and nuclei (DAPI, blue) in hMVECs. Representative examples are shown. Arrowheads indicate filopodia-like extrusions on CD34 + cells. Scale bar represents 25 µm (first 3 images) and 100 µm (last image). b HMVECs were analyzed for CD34 expression using flow cytometry. c – e Re-expression of CD34 in hMVECs after cell sorting. CD34 − cells (shown in c ) were cultured and CD34 expression was analyzed after 6 h ( d ) and 24 h ( e ). f, g The effect of exposure to VEGF or bFGF ( f ) and DLL4 ( g ) on the percentage of CD34 + tip cells. BSA was used as a control for DLL4. * p
    Figure Legend Snippet: Human microvascular endothelial cell cultures contain CD34 + tip cells. a Identification of tip cells by staining with anti-CD34 (green), F-actin (phalloidin, red), and nuclei (DAPI, blue) in hMVECs. Representative examples are shown. Arrowheads indicate filopodia-like extrusions on CD34 + cells. Scale bar represents 25 µm (first 3 images) and 100 µm (last image). b HMVECs were analyzed for CD34 expression using flow cytometry. c – e Re-expression of CD34 in hMVECs after cell sorting. CD34 − cells (shown in c ) were cultured and CD34 expression was analyzed after 6 h ( d ) and 24 h ( e ). f, g The effect of exposure to VEGF or bFGF ( f ) and DLL4 ( g ) on the percentage of CD34 + tip cells. BSA was used as a control for DLL4. * p

    Techniques Used: Staining, Expressing, Flow Cytometry, Cytometry, FACS, Cell Culture

    17) Product Images from "A regulatory microRNA network controls endothelial cell phenotypic switch during sprouting angiogenesis"

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

    Journal: eLife

    doi: 10.7554/eLife.48095

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

    Techniques Used: Migration, Generated, Transfection, Cell Culture

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

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

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

    Techniques Used: Western Blot, Concentration Assay

    18) Product Images from "WISP-1, a novel angiogenic regulator of the CCN family, promotes oral squamous cell carcinoma angiogenesis through VEGF-A expression"

    Article Title: WISP-1, a novel angiogenic regulator of the CCN family, promotes oral squamous cell carcinoma angiogenesis through VEGF-A expression

    Journal: Oncotarget

    doi:

    WISP-1 promotes VEGF-A expression in OSCC and contributing to angiogenesis through the HIF1-α signaling pathway (A) SCC4 cells were stimulated by WISP-1 (20 ng/mL) for the indicated times (0, 2, and 4 h). HIF1-α expression level was measured by western blot and qPCR. (B–E) SCC4 cells were pre-treated with HIF1-α inhibitor (1 μM) for 30 min or transfected with HIF1-α siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. The assay procedures were performed as described in Figure 3A–3D . (F) SCC4 cells were incubated with FAKi, PP2, AG1478, or U0126 for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 60 min. Chromatin immunoprecipitation (ChIP) assays were performed using an anti-HIF1-α antibody. One percent of the precipitated chromatin was analyzed to verify equal loading (input). Data are expressed as the mean ± SEM. * P
    Figure Legend Snippet: WISP-1 promotes VEGF-A expression in OSCC and contributing to angiogenesis through the HIF1-α signaling pathway (A) SCC4 cells were stimulated by WISP-1 (20 ng/mL) for the indicated times (0, 2, and 4 h). HIF1-α expression level was measured by western blot and qPCR. (B–E) SCC4 cells were pre-treated with HIF1-α inhibitor (1 μM) for 30 min or transfected with HIF1-α siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. The assay procedures were performed as described in Figure 3A–3D . (F) SCC4 cells were incubated with FAKi, PP2, AG1478, or U0126 for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 60 min. Chromatin immunoprecipitation (ChIP) assays were performed using an anti-HIF1-α antibody. One percent of the precipitated chromatin was analyzed to verify equal loading (input). Data are expressed as the mean ± SEM. * P

    Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Incubation, Chromatin Immunoprecipitation

    WISP-1 regulates the angiogenesis by raising VEGF-A expression in OSCC cells (A–B) SCC4 cells were incubated with WISP-1 (0–20 ng/mL) for 24 h, VEGF-A expression was measured by qPCR, ELISA, and western blot. (C–D) SCC4 cells were incubated with WISP-1 (0–20 ng/mL) for 24 h, and the CM was collected. EPCs were pre-treated for 30 min with IgG control antibody or VEGF-A antibody (1 μg/mL) and incubated with CM for 6 h and cell capillary-like structure formation in EPCs was examined by tube formation assay (C) EPCs were incubated with CM for 24 h, and cell migration was examined using the transwell assay (D) (E–F) SCC4 cells were incubated with the integrin αvβ3 antibody for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 24 h. VEGF-A expression was examined by western blot, qPCR, and ELISA. Data are expressed as mean ± SEM * P
    Figure Legend Snippet: WISP-1 regulates the angiogenesis by raising VEGF-A expression in OSCC cells (A–B) SCC4 cells were incubated with WISP-1 (0–20 ng/mL) for 24 h, VEGF-A expression was measured by qPCR, ELISA, and western blot. (C–D) SCC4 cells were incubated with WISP-1 (0–20 ng/mL) for 24 h, and the CM was collected. EPCs were pre-treated for 30 min with IgG control antibody or VEGF-A antibody (1 μg/mL) and incubated with CM for 6 h and cell capillary-like structure formation in EPCs was examined by tube formation assay (C) EPCs were incubated with CM for 24 h, and cell migration was examined using the transwell assay (D) (E–F) SCC4 cells were incubated with the integrin αvβ3 antibody for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 24 h. VEGF-A expression was examined by western blot, qPCR, and ELISA. Data are expressed as mean ± SEM * P

    Techniques Used: Expressing, Incubation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Tube Formation Assay, Migration, Transwell Assay

    FAK/Src signaling pathway is involved in WISP-1-promoted VEGF-A expression and contributing to angiogenesis (A–B) SCC4 cells were pre-treated with a FAK inhibitor (FAKi; 1 μM) for 30 min or transfected with FAK siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. VEGF-A expression was examined by western blot, qPCR, and ELISA. (C–D) SCC4 cells were pre-treated with a FAK inhibitor (FAKi; 1 μM), followed by WISP-1 (20 ng/mL) stimulation for 24 h. CM was collected. EPCs were incubated with CM for 6 h and capillary-like structure formation in EPCs was examined by tube formation assay (C) EPCs were incubated with CM for 24 h and cell migration was examined by transwell assay (D) (E–H) SCC4 cells were treated with a Src inhibitor (pp2; 1 μM) for 30 min or transfected with Src siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. Assay procedure was performed as in (A–D) (I) SCC4 cells were incubated with WISP-1 (20 ng/mL) for the indicated times, and FAK and c-Src phosphorylation was determined by western blot. (J–K) SCC4 cells were incubated with an integrin αvβ3 antibody or FAKi for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 60 min, and FAK (J) and c-Src (K) phosphorylation was determined by western blot. Data are expressed as the mean ± SEM * P
    Figure Legend Snippet: FAK/Src signaling pathway is involved in WISP-1-promoted VEGF-A expression and contributing to angiogenesis (A–B) SCC4 cells were pre-treated with a FAK inhibitor (FAKi; 1 μM) for 30 min or transfected with FAK siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. VEGF-A expression was examined by western blot, qPCR, and ELISA. (C–D) SCC4 cells were pre-treated with a FAK inhibitor (FAKi; 1 μM), followed by WISP-1 (20 ng/mL) stimulation for 24 h. CM was collected. EPCs were incubated with CM for 6 h and capillary-like structure formation in EPCs was examined by tube formation assay (C) EPCs were incubated with CM for 24 h and cell migration was examined by transwell assay (D) (E–H) SCC4 cells were treated with a Src inhibitor (pp2; 1 μM) for 30 min or transfected with Src siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. Assay procedure was performed as in (A–D) (I) SCC4 cells were incubated with WISP-1 (20 ng/mL) for the indicated times, and FAK and c-Src phosphorylation was determined by western blot. (J–K) SCC4 cells were incubated with an integrin αvβ3 antibody or FAKi for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 60 min, and FAK (J) and c-Src (K) phosphorylation was determined by western blot. Data are expressed as the mean ± SEM * P

    Techniques Used: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Incubation, Tube Formation Assay, Migration, Transwell Assay

    EGFR transactivation is involved in WISP-1-induced VEGF-A expression and contributing to angiogenesis (A–D) SCC4 cells were pre-treated with an EGFR inhibitor (AG1478; 1 μM) for 30 min or transfected with EGFR siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. The assay procedures were performed as described in Figure 3A–3D . (E–H) SCC4 cells were treated by an ERK inhibitor (U0126; 1 μM) for 30 min or transfected with ERK siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. The assay procedures were performed as described in Figure 3A–3D . (I) SCC4 cells were incubated with WISP-1 (20 ng/mL) for the indicated times and EGFR and ERK phosphorylation was determined by western blot. (J–K) SCC4 cells were incubated with the integrin αvβ3 antibody, FAKi, PP2, or AG1478 for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 60 min, and EGFR (J) and ERK (K) phosphorylation was determined by western blot. Data are expressed as the mean ± SEM * P
    Figure Legend Snippet: EGFR transactivation is involved in WISP-1-induced VEGF-A expression and contributing to angiogenesis (A–D) SCC4 cells were pre-treated with an EGFR inhibitor (AG1478; 1 μM) for 30 min or transfected with EGFR siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. The assay procedures were performed as described in Figure 3A–3D . (E–H) SCC4 cells were treated by an ERK inhibitor (U0126; 1 μM) for 30 min or transfected with ERK siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. The assay procedures were performed as described in Figure 3A–3D . (I) SCC4 cells were incubated with WISP-1 (20 ng/mL) for the indicated times and EGFR and ERK phosphorylation was determined by western blot. (J–K) SCC4 cells were incubated with the integrin αvβ3 antibody, FAKi, PP2, or AG1478 for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 60 min, and EGFR (J) and ERK (K) phosphorylation was determined by western blot. Data are expressed as the mean ± SEM * P

    Techniques Used: Expressing, Transfection, Incubation, Western Blot

    WISP-1 knockdown in OSCC decreases VEGF-A expression and angiogenesis-related tumor growth in vivo (A) SCC4 cells stably expressing shRNA constructs or control shRNA were seeded as monolayers and counted daily. Cells (10 3 ) were plated in 6 well plates and grown for 2 days. Cells were trypsinized, and cell numbers was counted. (B–C) WISP-1 and VEGF-A mRNA and protein expression in SCC4 cells stably expressed a control shRNA or a WISP-1 shRNA was examined by western blot, qPCR, and ELISA. (D–E) EPCs were incubated with CM collected from control-shRNA and WISP-1-shRNA transfected SCC4 cells for 24 h and cell migration or tube formation were examined. (F) PBS, VEGF-A, control shRNA/SCC4 CM, and WISP-1 shRNA/SCC4 CM mixed in Matrigel were placed on chick chorioallantoic membranes. CAMs in each group were photographed on developmental day 12. (G) Mice were subcutaneously injected with Matrigel mixed with PBS, control shRNA/SCC4 CM or WISP-1 shRNA/SCC4 CM for seven days. Plugs excised from the mice were photographed and stained with CD31. (H) Control shRNA and WISP-1 shRNA SCC4 cells were mixed with Matrigel and injected into the flank of the mice for 28 days. Tumor growth was monitored using the IVIS Imaging System. Tumor growth was quantified by fluorescent imaging from week 0–6. (I) Tumors were paraffin embedded, and sections were immunostained using the WISP-1, VEGF-A, and CD31 antibodies. (E = epithelial, T = tumor, S = stroma). (J) Diagrammatic model for the role of WISP-1 in OSCC. (1) WISP-1 induces VEGF-A expression and secretion in OSCC cells through the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signal pathway. (2) The WISP-1-induced secretion of VEGF-A subsequently recruiting EPCs to OSCC tumor microenvironment and promoting neoangiogenesis. Data represent the mean ± SEM * P
    Figure Legend Snippet: WISP-1 knockdown in OSCC decreases VEGF-A expression and angiogenesis-related tumor growth in vivo (A) SCC4 cells stably expressing shRNA constructs or control shRNA were seeded as monolayers and counted daily. Cells (10 3 ) were plated in 6 well plates and grown for 2 days. Cells were trypsinized, and cell numbers was counted. (B–C) WISP-1 and VEGF-A mRNA and protein expression in SCC4 cells stably expressed a control shRNA or a WISP-1 shRNA was examined by western blot, qPCR, and ELISA. (D–E) EPCs were incubated with CM collected from control-shRNA and WISP-1-shRNA transfected SCC4 cells for 24 h and cell migration or tube formation were examined. (F) PBS, VEGF-A, control shRNA/SCC4 CM, and WISP-1 shRNA/SCC4 CM mixed in Matrigel were placed on chick chorioallantoic membranes. CAMs in each group were photographed on developmental day 12. (G) Mice were subcutaneously injected with Matrigel mixed with PBS, control shRNA/SCC4 CM or WISP-1 shRNA/SCC4 CM for seven days. Plugs excised from the mice were photographed and stained with CD31. (H) Control shRNA and WISP-1 shRNA SCC4 cells were mixed with Matrigel and injected into the flank of the mice for 28 days. Tumor growth was monitored using the IVIS Imaging System. Tumor growth was quantified by fluorescent imaging from week 0–6. (I) Tumors were paraffin embedded, and sections were immunostained using the WISP-1, VEGF-A, and CD31 antibodies. (E = epithelial, T = tumor, S = stroma). (J) Diagrammatic model for the role of WISP-1 in OSCC. (1) WISP-1 induces VEGF-A expression and secretion in OSCC cells through the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signal pathway. (2) The WISP-1-induced secretion of VEGF-A subsequently recruiting EPCs to OSCC tumor microenvironment and promoting neoangiogenesis. Data represent the mean ± SEM * P

    Techniques Used: Expressing, In Vivo, Stable Transfection, shRNA, Construct, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Incubation, Transfection, Migration, Mouse Assay, Injection, Staining, Imaging

    Clinical significance of WISP-1 and VEGF-A in specimens from patients with OSCC Tumor specimens were immunostained (IHC) with anti-WISP-1 and anti-VEGF-A antibodies. The staining intensity was scored 1–5. (A) IHC photographs. (E = epithelial, T = tumor, S = stroma). (B–D) Quantitative results and correlation between WISP-1, VEGF-A, and OSCC clinical grade.
    Figure Legend Snippet: Clinical significance of WISP-1 and VEGF-A in specimens from patients with OSCC Tumor specimens were immunostained (IHC) with anti-WISP-1 and anti-VEGF-A antibodies. The staining intensity was scored 1–5. (A) IHC photographs. (E = epithelial, T = tumor, S = stroma). (B–D) Quantitative results and correlation between WISP-1, VEGF-A, and OSCC clinical grade.

    Techniques Used: Immunohistochemistry, Staining

    19) Product Images from "Genetic perturbation of IFN-α transcriptional modulators in human endothelial cells uncovers pivotal regulators of angiogenesis"

    Article Title: Genetic perturbation of IFN-α transcriptional modulators in human endothelial cells uncovers pivotal regulators of angiogenesis

    Journal: Computational and Structural Biotechnology Journal

    doi: 10.1016/j.csbj.2020.11.048

    Modulation of STAT1, USP18, IFIH1, GBP1, and IRF1 affects in vitro EC sprouting. (a) Representative pictures of spheroids obtained from 4 independent sprouting assays on HUVECs treated with 20 ng/ml VEGF, with or without 600 IU/ml IFN-α, upon STAT1, USP18, IFIH1, GBP1 and IRF1 silencing or scrambled siRNA treatment (siCTRL). (b) Quantification of sprout areas from 4 independent sprouting assays in the absence and in the presence of IFN-α. *** p
    Figure Legend Snippet: Modulation of STAT1, USP18, IFIH1, GBP1, and IRF1 affects in vitro EC sprouting. (a) Representative pictures of spheroids obtained from 4 independent sprouting assays on HUVECs treated with 20 ng/ml VEGF, with or without 600 IU/ml IFN-α, upon STAT1, USP18, IFIH1, GBP1 and IRF1 silencing or scrambled siRNA treatment (siCTRL). (b) Quantification of sprout areas from 4 independent sprouting assays in the absence and in the presence of IFN-α. *** p

    Techniques Used: In Vitro

    20) Product Images from "PPemd26, an anthraquinone derivative, suppresses angiogenesis via inhibiting VEGFR2 signalling"

    Article Title: PPemd26, an anthraquinone derivative, suppresses angiogenesis via inhibiting VEGFR2 signalling

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.12872

    PPemd26 inhibits VEGF-A-induced proliferation of HUVECs. (A) After starvation, cells were pretreated with different PPemd compounds at 10 μM followed by the stimulation with VEGF-A (25 ng·mL −1 ) for another 24 h. Cell viability was then determined by MTT assay. Each column represents the mean ± SEM of three independent experiments performed in duplicate. (B) Chemical structure of PPemd 26. (C) After starvation, cells were pretreated with indicated concentrations of PPemd26 or sunitinib followed by the stimulation with VEGF-A (25 ng·mL −1 ) for another 24 h. Cell viability was then determined by MTT assay. Each column represents the mean ± SEM of at least three independent experiments performed in duplicate. (D) HUVECs were treated as in (C), and cell proliferation was determined. Each column represents the mean ± SEM of five independent experiments performed in duplicate. (E) HUVECs were treated as in (C), and cytotoxicity of PPemd26 was determined by LDH assay. Cells were also treated with cell lysis buffer (total lysis, TL) to serve as positive control. * P
    Figure Legend Snippet: PPemd26 inhibits VEGF-A-induced proliferation of HUVECs. (A) After starvation, cells were pretreated with different PPemd compounds at 10 μM followed by the stimulation with VEGF-A (25 ng·mL −1 ) for another 24 h. Cell viability was then determined by MTT assay. Each column represents the mean ± SEM of three independent experiments performed in duplicate. (B) Chemical structure of PPemd 26. (C) After starvation, cells were pretreated with indicated concentrations of PPemd26 or sunitinib followed by the stimulation with VEGF-A (25 ng·mL −1 ) for another 24 h. Cell viability was then determined by MTT assay. Each column represents the mean ± SEM of at least three independent experiments performed in duplicate. (D) HUVECs were treated as in (C), and cell proliferation was determined. Each column represents the mean ± SEM of five independent experiments performed in duplicate. (E) HUVECs were treated as in (C), and cytotoxicity of PPemd26 was determined by LDH assay. Cells were also treated with cell lysis buffer (total lysis, TL) to serve as positive control. * P

    Techniques Used: MTT Assay, Lactate Dehydrogenase Assay, Lysis, Positive Control

    PPemd 26 inhibits VEGFR2-mediated signalling. Cells were pretreated with indicated concentrations of PPemd26 for 30 min, followed by the addition of VEGF-A (25 ng·mL −1 ) for another 5 min (A) or 30 min (B–E). Phosphorylation status of VEGFR2 (A), Akt (B), ERK1/2 (C), FAK (D) and Src (E) was determined by immunoblotting. Bar graphs represent the mean ± SEM of at least four independent experiments. * P
    Figure Legend Snippet: PPemd 26 inhibits VEGFR2-mediated signalling. Cells were pretreated with indicated concentrations of PPemd26 for 30 min, followed by the addition of VEGF-A (25 ng·mL −1 ) for another 5 min (A) or 30 min (B–E). Phosphorylation status of VEGFR2 (A), Akt (B), ERK1/2 (C), FAK (D) and Src (E) was determined by immunoblotting. Bar graphs represent the mean ± SEM of at least four independent experiments. * P

    Techniques Used:

    PPemd 26 inhibits VEGF-A- or tumour-induced neovascularization. (A) Rat aortic rings were treated with various concentrations of PPemd26 in the presence or absence of VEGF-A (25 ng·mL −1 ). The formation of vessel sprouts from aortic rings was examined on day 8. Bar graphs show compiled data of average microvessel area ( n = 7). * P
    Figure Legend Snippet: PPemd 26 inhibits VEGF-A- or tumour-induced neovascularization. (A) Rat aortic rings were treated with various concentrations of PPemd26 in the presence or absence of VEGF-A (25 ng·mL −1 ). The formation of vessel sprouts from aortic rings was examined on day 8. Bar graphs show compiled data of average microvessel area ( n = 7). * P

    Techniques Used:

    Sunitinib inhibits VEGF-A-induced invasion and tube formation in HUVECs. (A) Cells were starved and seeded in the absence or presence of sunitinib using VEGF-A as the chemoattractant. After 16 h, invading cells were stained and quantified. (B) HUVECs were seeded on Matrigel in the presence of VEGF-A with or without sunitinib at indicated concentrations. Cells were photographed under phase contrast after 16 h. Bar graphs show compiled data of average rate of invading cell numbers (A) ( n = 4) and average sprout arch numbers (B) ( n = 4). * P
    Figure Legend Snippet: Sunitinib inhibits VEGF-A-induced invasion and tube formation in HUVECs. (A) Cells were starved and seeded in the absence or presence of sunitinib using VEGF-A as the chemoattractant. After 16 h, invading cells were stained and quantified. (B) HUVECs were seeded on Matrigel in the presence of VEGF-A with or without sunitinib at indicated concentrations. Cells were photographed under phase contrast after 16 h. Bar graphs show compiled data of average rate of invading cell numbers (A) ( n = 4) and average sprout arch numbers (B) ( n = 4). * P

    Techniques Used: Staining

    PPemd 26 inhibits VEGF-A-induced migration, invasion and tube formation in HUVECs. (A) After starvation, cell monolayers were scratched and treated with vehicle or indicated concentrations of PPemd26 in the presence of VEGF-A for another 24 h. The rate of cell migration into the scratch was then determined. (B) Cells were starved and seeded in the absence or presence of PPemd26 using VEGF-A as the chemoattractant. After 16 h, invading cells were stained and quantified. (C) HUVECs were seeded on Matrigel in the presence of VEGF-A with or without PPemd26 at indicated concentrations. Cells were photographed under phase contrast after 16 h. Bar graphs show compiled data of average rate of cell migration (A) ( n = 4), invading cell numbers (B) ( n = 3), and average sprout arch numbers (C) ( n = 8). * P
    Figure Legend Snippet: PPemd 26 inhibits VEGF-A-induced migration, invasion and tube formation in HUVECs. (A) After starvation, cell monolayers were scratched and treated with vehicle or indicated concentrations of PPemd26 in the presence of VEGF-A for another 24 h. The rate of cell migration into the scratch was then determined. (B) Cells were starved and seeded in the absence or presence of PPemd26 using VEGF-A as the chemoattractant. After 16 h, invading cells were stained and quantified. (C) HUVECs were seeded on Matrigel in the presence of VEGF-A with or without PPemd26 at indicated concentrations. Cells were photographed under phase contrast after 16 h. Bar graphs show compiled data of average rate of cell migration (A) ( n = 4), invading cell numbers (B) ( n = 3), and average sprout arch numbers (C) ( n = 8). * P

    Techniques Used: Migration, Staining

    21) Product Images from "IGF2 and IGF1R identified as novel tip cell genes in primary microvascular endothelial cell monolayers"

    Article Title: IGF2 and IGF1R identified as novel tip cell genes in primary microvascular endothelial cell monolayers

    Journal: Angiogenesis

    doi: 10.1007/s10456-018-9627-4

    IGF2 and IGF1R are essential for CD34 + tip cell fate. a, b Effect of knockdown of IGF2 and IGF1R expression on percentages of CD34 + tip cells. Bars show percentages of CD34 + hMVECs ( a ) and HUVECs ( b ) treated with siNT, siIGF2 , or siIGF1R as detected by flow cytometry. c, d Quantification of numbers of sprouts ( c ) and average sprout length ( d ) of spheroids composed of HUVECs after treatment with siNT, siIGF2 , or siIGF1R . e Analysis of CD34 + tip cell morphology after knockdown of IGF2 and IGF1R expression. Staining of CD34 (green), F-actin (phalloidin, red), and nuclei (DAPI, blue) in hMVECs. Scale bars represent 50 µm (first 3 columns) and 100 µm (last column). f Effect of rhIGF2 on CD34 + HUVEC tip cell percentages. Bars shows CD34 + tip cells of HUVECs treated with either 25 ng/mL BSA, 25 ng/mL VEGF-A, or 50 ng/mL rhIGF2 as detected by flow cytometry. g, h Effects of VEGF and IGF2 on the number of sprouts ( g ) and average sprout length ( h ) in spheroids of CD34 + tip cells or CD34 − non-tip cells. Data in a – d and f – h are shown as mean ± standard deviation after factor correction. * p
    Figure Legend Snippet: IGF2 and IGF1R are essential for CD34 + tip cell fate. a, b Effect of knockdown of IGF2 and IGF1R expression on percentages of CD34 + tip cells. Bars show percentages of CD34 + hMVECs ( a ) and HUVECs ( b ) treated with siNT, siIGF2 , or siIGF1R as detected by flow cytometry. c, d Quantification of numbers of sprouts ( c ) and average sprout length ( d ) of spheroids composed of HUVECs after treatment with siNT, siIGF2 , or siIGF1R . e Analysis of CD34 + tip cell morphology after knockdown of IGF2 and IGF1R expression. Staining of CD34 (green), F-actin (phalloidin, red), and nuclei (DAPI, blue) in hMVECs. Scale bars represent 50 µm (first 3 columns) and 100 µm (last column). f Effect of rhIGF2 on CD34 + HUVEC tip cell percentages. Bars shows CD34 + tip cells of HUVECs treated with either 25 ng/mL BSA, 25 ng/mL VEGF-A, or 50 ng/mL rhIGF2 as detected by flow cytometry. g, h Effects of VEGF and IGF2 on the number of sprouts ( g ) and average sprout length ( h ) in spheroids of CD34 + tip cells or CD34 − non-tip cells. Data in a – d and f – h are shown as mean ± standard deviation after factor correction. * p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Standard Deviation

    Human microvascular endothelial cell cultures contain CD34 + tip cells. a Identification of tip cells by staining with anti-CD34 (green), F-actin (phalloidin, red), and nuclei (DAPI, blue) in hMVECs. Representative examples are shown. Arrowheads indicate filopodia-like extrusions on CD34 + cells. Scale bar represents 25 µm (first 3 images) and 100 µm (last image). b HMVECs were analyzed for CD34 expression using flow cytometry. c – e Re-expression of CD34 in hMVECs after cell sorting. CD34 − cells (shown in c ) were cultured and CD34 expression was analyzed after 6 h ( d ) and 24 h ( e ). f, g The effect of exposure to VEGF or bFGF ( f ) and DLL4 ( g ) on the percentage of CD34 + tip cells. BSA was used as a control for DLL4. * p
    Figure Legend Snippet: Human microvascular endothelial cell cultures contain CD34 + tip cells. a Identification of tip cells by staining with anti-CD34 (green), F-actin (phalloidin, red), and nuclei (DAPI, blue) in hMVECs. Representative examples are shown. Arrowheads indicate filopodia-like extrusions on CD34 + cells. Scale bar represents 25 µm (first 3 images) and 100 µm (last image). b HMVECs were analyzed for CD34 expression using flow cytometry. c – e Re-expression of CD34 in hMVECs after cell sorting. CD34 − cells (shown in c ) were cultured and CD34 expression was analyzed after 6 h ( d ) and 24 h ( e ). f, g The effect of exposure to VEGF or bFGF ( f ) and DLL4 ( g ) on the percentage of CD34 + tip cells. BSA was used as a control for DLL4. * p

    Techniques Used: Staining, Expressing, Flow Cytometry, Cytometry, FACS, Cell Culture

    22) Product Images from "Endothelial NO Synthase-Dependent S-Nitrosylation of β-Catenin Prevents Its Association with TCF4 and Inhibits Proliferation of Endothelial Cells Stimulated by Wnt3a"

    Article Title: Endothelial NO Synthase-Dependent S-Nitrosylation of β-Catenin Prevents Its Association with TCF4 and Inhibits Proliferation of Endothelial Cells Stimulated by Wnt3a

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00089-17

    VEGF inhibits Wnt/β-catenin signaling in an eNOS-dependent manner. (A) qRT-PCR analysis of axin2 mRNA levels in control or eNOS-depleted BAECs treated with Wnt3a-conditioned medium and/or VEGF (40 ng/ml; 24 h; n = 3) as indicated. eNOS was depleted in BAECs by transfection of siRNA against eNOS (eNOS-siRNA), and CT-siRNA was used for comparison. Depletion of eNOS was monitored by IB, and β-actin was used as a loading control. (B) BrdU incorporation assay in BAECs treated with Wnt3a-conditioned medium and/or VEGF (40 ng/ml; 24 h) as indicated. The percentage of BrdU-positive cells for each treatment was normalized to that of nontreated cells ( n = 3). (C) qRT-PCR analysis of cyclin D1 mRNA levels in BAECs treated with Wnt3a-conditioned medium and/or VEGF (40 ng/ml; 6 h; n = 3) as indicated. The data are represented as means and SEM. *, P
    Figure Legend Snippet: VEGF inhibits Wnt/β-catenin signaling in an eNOS-dependent manner. (A) qRT-PCR analysis of axin2 mRNA levels in control or eNOS-depleted BAECs treated with Wnt3a-conditioned medium and/or VEGF (40 ng/ml; 24 h; n = 3) as indicated. eNOS was depleted in BAECs by transfection of siRNA against eNOS (eNOS-siRNA), and CT-siRNA was used for comparison. Depletion of eNOS was monitored by IB, and β-actin was used as a loading control. (B) BrdU incorporation assay in BAECs treated with Wnt3a-conditioned medium and/or VEGF (40 ng/ml; 24 h) as indicated. The percentage of BrdU-positive cells for each treatment was normalized to that of nontreated cells ( n = 3). (C) qRT-PCR analysis of cyclin D1 mRNA levels in BAECs treated with Wnt3a-conditioned medium and/or VEGF (40 ng/ml; 6 h; n = 3) as indicated. The data are represented as means and SEM. *, P

    Techniques Used: Quantitative RT-PCR, Transfection, BrdU Incorporation Assay

    23) Product Images from "VEGF-C Alters Barrier Function of Cultured Lymphatic Endothelial Cells Through a VEGFR-3-Dependent Mechanism"

    Article Title: VEGF-C Alters Barrier Function of Cultured Lymphatic Endothelial Cells Through a VEGFR-3-Dependent Mechanism

    Journal: Lymphatic research and biology

    doi: 10.1089/lrb.2007.1004

    Inhibition of VEGFR-3 blocks VEGF-C156S-induced changes in TER in HMLEC-d. MAZ51 (5 μM) was added 30 minutes prior to VEGF-C156S (10 nM), and itself did not alter TER. However, MAZ51-treated HMLEC-d did not display decreased TER after treatment with VEGF-C156S, unlike cells that were not treated with MAZ51. The tracings show the average TER from both groups. N=4 for both groups.
    Figure Legend Snippet: Inhibition of VEGFR-3 blocks VEGF-C156S-induced changes in TER in HMLEC-d. MAZ51 (5 μM) was added 30 minutes prior to VEGF-C156S (10 nM), and itself did not alter TER. However, MAZ51-treated HMLEC-d did not display decreased TER after treatment with VEGF-C156S, unlike cells that were not treated with MAZ51. The tracings show the average TER from both groups. N=4 for both groups.

    Techniques Used: Inhibition

    VEGF-A alters barrier function in HUVEC, but not in HMLEC-d. A . Tracings of the time-courses of HUVEC and HMLEC-d TER before and during exposure to 1 nM VEGF-A are shown. The tracings represent the averages of N=4 wells each. Panel B shows a comparison of the mean fractional change in TER 1 min. after VEGF-A was added. **P
    Figure Legend Snippet: VEGF-A alters barrier function in HUVEC, but not in HMLEC-d. A . Tracings of the time-courses of HUVEC and HMLEC-d TER before and during exposure to 1 nM VEGF-A are shown. The tracings represent the averages of N=4 wells each. Panel B shows a comparison of the mean fractional change in TER 1 min. after VEGF-A was added. **P

    Techniques Used:

    24) Product Images from "Intracrine Vascular Endothelial Growth Factor Signaling in Survival and Chemoresistance of Human Colorectal Cancer Cells"

    Article Title: Intracrine Vascular Endothelial Growth Factor Signaling in Survival and Chemoresistance of Human Colorectal Cancer Cells

    Journal: Oncogene

    doi: 10.1038/onc.2010.496

    Effect of loss of VEGF expression on viability of CRC cells in-vitro A. Loss of VEGF expression led to increased spontaneous cell death. Cells were grown in 1% FBS medium for 48h, fixed and stained with PI. Cell death was assessed by flow cytometry. B. Increased spontaneous apoptosis in HCT116 VEGF −/− cells. Cells were grown in 1% FBS medium for 48h, and apoptosis was assessed by flow cytometry following Annexin V staining. C. Altered expression of apoptotic mediators in VEGF −/− cells. Whole-cell lysates of cells growing in 1% FBS medium for 48h were collected and analyzed for expression of caspase 3, cleaved caspase-3, bax and survivin by western blot analysis. Actin served as a loading control.
    Figure Legend Snippet: Effect of loss of VEGF expression on viability of CRC cells in-vitro A. Loss of VEGF expression led to increased spontaneous cell death. Cells were grown in 1% FBS medium for 48h, fixed and stained with PI. Cell death was assessed by flow cytometry. B. Increased spontaneous apoptosis in HCT116 VEGF −/− cells. Cells were grown in 1% FBS medium for 48h, and apoptosis was assessed by flow cytometry following Annexin V staining. C. Altered expression of apoptotic mediators in VEGF −/− cells. Whole-cell lysates of cells growing in 1% FBS medium for 48h were collected and analyzed for expression of caspase 3, cleaved caspase-3, bax and survivin by western blot analysis. Actin served as a loading control.

    Techniques Used: Expressing, In Vitro, Staining, Flow Cytometry, Cytometry, Western Blot

    Effect of loss of VEGF expression on proliferation of CRC cells A. Loss of VEGF expression in CRC cells with deletion of VEGF alleles. VEGF-A levels in conditioned medium from HCT116 and LS174T VEGF +/+ and VEGF −/− cells were determined by western blot analysis. VEGF-A expression was undetectable in the VEGF −/− cells. Equalproteinloading of the gels was verified by Ponseu-S staining of the membranes. B. The growth rates of HCT116 and LS174T VEGF +/+ and VEGF −/− cells were assessed in terms of absorbance at 570 nm in an MTT assay. VEGF −/− cells showed significantly reduced proliferative activity compared with VEGF +/+ cells.
    Figure Legend Snippet: Effect of loss of VEGF expression on proliferation of CRC cells A. Loss of VEGF expression in CRC cells with deletion of VEGF alleles. VEGF-A levels in conditioned medium from HCT116 and LS174T VEGF +/+ and VEGF −/− cells were determined by western blot analysis. VEGF-A expression was undetectable in the VEGF −/− cells. Equalproteinloading of the gels was verified by Ponseu-S staining of the membranes. B. The growth rates of HCT116 and LS174T VEGF +/+ and VEGF −/− cells were assessed in terms of absorbance at 570 nm in an MTT assay. VEGF −/− cells showed significantly reduced proliferative activity compared with VEGF +/+ cells.

    Techniques Used: Expressing, Western Blot, Staining, MTT Assay, Activity Assay

    Effect of loss of VEGF expression on chemosensitivity and signaling in CRC cells A. Increased chemosensitivity of VEGF −/− cells to 5FU treatment. HCT116 and LS174T VEGF +/+ and VEGF −/− cells growing in 1% FBS medium were treated without or with 5FU for 48h, fixed and stained with PI; cell death was then assessed by flow cytometry. B. 5FU treatment led to increased apoptosis in HCT116 VEGF −/− cells. HCT116 VEGF +/+ and VEGF −/− cells growing in 1% FBS medium were treated without or with 5FU for 48h, then stained with Annexin V; apoptosis was then assessed by flow cytometry. C. 5FU treatment led to increased expression of proapoptotic mediators in HCT116 and LS174T VEGF −/− cells. Whole-cell lysates were collected from cells treated without or with 5FU for 48h and were analyzed for expression of caspase-3, cleaved caspase-3, PARP and cleaved PARP by western blot analysis. Vinculin served as a loading control. D. 5FU treatment led to increased PARP cleavage in HCT116 VEGF −/− cells in a dose-dependent manner. Whole-cell lysates were collected from HCT116 VEGF +/+ and VEGF −/− cells treated without or with increasing concentrations of 5FU for 48h; the lysates were analyzed for expression of PARP and cleaved PARP by western blot analysis. The bands for cleaved PARP were scanned, densitometrically quantitated using NIH Image J software and the resulting data wer e plotted (right panel.)
    Figure Legend Snippet: Effect of loss of VEGF expression on chemosensitivity and signaling in CRC cells A. Increased chemosensitivity of VEGF −/− cells to 5FU treatment. HCT116 and LS174T VEGF +/+ and VEGF −/− cells growing in 1% FBS medium were treated without or with 5FU for 48h, fixed and stained with PI; cell death was then assessed by flow cytometry. B. 5FU treatment led to increased apoptosis in HCT116 VEGF −/− cells. HCT116 VEGF +/+ and VEGF −/− cells growing in 1% FBS medium were treated without or with 5FU for 48h, then stained with Annexin V; apoptosis was then assessed by flow cytometry. C. 5FU treatment led to increased expression of proapoptotic mediators in HCT116 and LS174T VEGF −/− cells. Whole-cell lysates were collected from cells treated without or with 5FU for 48h and were analyzed for expression of caspase-3, cleaved caspase-3, PARP and cleaved PARP by western blot analysis. Vinculin served as a loading control. D. 5FU treatment led to increased PARP cleavage in HCT116 VEGF −/− cells in a dose-dependent manner. Whole-cell lysates were collected from HCT116 VEGF +/+ and VEGF −/− cells treated without or with increasing concentrations of 5FU for 48h; the lysates were analyzed for expression of PARP and cleaved PARP by western blot analysis. The bands for cleaved PARP were scanned, densitometrically quantitated using NIH Image J software and the resulting data wer e plotted (right panel.)

    Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry, Western Blot, Software

    25) Product Images from "Triphala and Its Active Constituent Chebulinic Acid Are Natural Inhibitors of Vascular Endothelial Growth Factor-A Mediated Angiogenesis"

    Article Title: Triphala and Its Active Constituent Chebulinic Acid Are Natural Inhibitors of Vascular Endothelial Growth Factor-A Mediated Angiogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043934

    The effects of Triphala churna (THL) and chebulinic acid (CI) on vascular endothelial growth factor (VEGF) induced tube formation and permeability in human umbilical vein cells (HUVEC). In contrast to ( A ) control, ( B ) VEGF promotes tube formation in HUVEC. However, treatment with either ( C ) THL or ( D ) CI inhibits VEGF induced tube formation in HUVEC. ( E ) Similarly, VEGF induces significant permeability in HUVEC in comparison to untreated control (+, p
    Figure Legend Snippet: The effects of Triphala churna (THL) and chebulinic acid (CI) on vascular endothelial growth factor (VEGF) induced tube formation and permeability in human umbilical vein cells (HUVEC). In contrast to ( A ) control, ( B ) VEGF promotes tube formation in HUVEC. However, treatment with either ( C ) THL or ( D ) CI inhibits VEGF induced tube formation in HUVEC. ( E ) Similarly, VEGF induces significant permeability in HUVEC in comparison to untreated control (+, p

    Techniques Used: Permeability

    The effects of THL (triphala churna) on in vivo matrigel angiogenesis assay. ( A ) Photographs of representative matrigel plugs show THL untreated red colored vascular endothelial growth factor (VEGF) containing plugs in comparison to the VEGF minus PBS containing controls. ( A ) In contrast, THL (triphala churna) treated VEGF containing matrigel plugs were pale. ( B ) Masson's trichrome staining (endothelial cells stain red and matrigel stains blue) and ( C, D ) CD31 immunohistochemistry of the matrigel plug sections show large numbers of endothelial cells in THL untreated VEGF containing plugs in comparison to controls (+, p
    Figure Legend Snippet: The effects of THL (triphala churna) on in vivo matrigel angiogenesis assay. ( A ) Photographs of representative matrigel plugs show THL untreated red colored vascular endothelial growth factor (VEGF) containing plugs in comparison to the VEGF minus PBS containing controls. ( A ) In contrast, THL (triphala churna) treated VEGF containing matrigel plugs were pale. ( B ) Masson's trichrome staining (endothelial cells stain red and matrigel stains blue) and ( C, D ) CD31 immunohistochemistry of the matrigel plug sections show large numbers of endothelial cells in THL untreated VEGF containing plugs in comparison to controls (+, p

    Techniques Used: In Vivo, Angiogenesis Assay, Staining, Immunohistochemistry

    The effects of THL (triphala churna) and chebulinic acid (CI) on vascular endothelial growth factor (VEGF) induced migration of human umbilical vein cells (HUVEC). Phase-contrast microphotographs of the wound area in HUVEC monolayer at 18 h after wounding ( A–D ) VEGF promotes complete wound closure or healing in 18vh. In contrast, this effect is lost when cells are treated either with THL ( A, C ) or CI ( B, D ). Wound healing is calculated as the distance covered by cells in relation to the initial wound distance at 0 h and is expressed as a percentage of the initial distance at 0 h. *, P
    Figure Legend Snippet: The effects of THL (triphala churna) and chebulinic acid (CI) on vascular endothelial growth factor (VEGF) induced migration of human umbilical vein cells (HUVEC). Phase-contrast microphotographs of the wound area in HUVEC monolayer at 18 h after wounding ( A–D ) VEGF promotes complete wound closure or healing in 18vh. In contrast, this effect is lost when cells are treated either with THL ( A, C ) or CI ( B, D ). Wound healing is calculated as the distance covered by cells in relation to the initial wound distance at 0 h and is expressed as a percentage of the initial distance at 0 h. *, P

    Techniques Used: Migration

    The effects of Triphala churna (THL) and chebulinic acid (CI) on vascular endothelial growth factor (VEGF) induced angiogenesis in the CAM assay. ( A, E ) PBS used as a control does not induce blood vessel formation. ( B, E ) VEGF induces new blood vessel formation. ( C, E ) THL inhibits VEGF induced new blood vessel formation ( D, E ) CI inhibits VEGF mediated new blood vessel formation. Representative photographs of six separate experiments are shown.
    Figure Legend Snippet: The effects of Triphala churna (THL) and chebulinic acid (CI) on vascular endothelial growth factor (VEGF) induced angiogenesis in the CAM assay. ( A, E ) PBS used as a control does not induce blood vessel formation. ( B, E ) VEGF induces new blood vessel formation. ( C, E ) THL inhibits VEGF induced new blood vessel formation ( D, E ) CI inhibits VEGF mediated new blood vessel formation. Representative photographs of six separate experiments are shown.

    Techniques Used: Chick Chorioallantoic Membrane Assay

    26) Product Images from "Simultaneous in vivo imaging of blood and lymphatic vessel growth in Prox1-GFP/Flk1::myr-mCherry mice"

    Article Title: Simultaneous in vivo imaging of blood and lymphatic vessel growth in Prox1-GFP/Flk1::myr-mCherry mice

    Journal: The FEBS journal

    doi: 10.1111/febs.13234

    VEGF pellet implantation induces blood and lymphatic vessel formation In vivo observation of angiogenesis and lymphangiogenesis in a Prox1-GFP/Flk1::myr-mCherry mouse over 10 days following 150ng VEGF pellet implantation (left 3 columns) or control PBS pellet implantation (far right column). ( A-P ) SteREO Lumar microscopy images of GFP-expressing lymphatic vessels (green), mCherry-expressing blood vessels (red) and overlays (right two columns): ( A-D ) prior to implantation, ( E-H ) 3 days post-implantation, ( I-L ) 7 days post-implantation, and ( MP ) 10 days post-implantation. ( Q-T ) Confocal imaging of the same corneas on day 10. Scale bar: 500 μm in ( A-P ) and 200 μm in ( Q-T ). Arrows ( A ): regularly spaced lymphatic vessels penetrating the cornea in the uninjured eye; arrowheads ( E ): new lymphatic vessels budding from the cornea 3 days after VEGF pellet implantation; asterisk ( I ): one potential new lymphatic vessel budding from the cornea on day 7 after the initial phase; circle ( T ): outline of the implanted PBS control pellet.
    Figure Legend Snippet: VEGF pellet implantation induces blood and lymphatic vessel formation In vivo observation of angiogenesis and lymphangiogenesis in a Prox1-GFP/Flk1::myr-mCherry mouse over 10 days following 150ng VEGF pellet implantation (left 3 columns) or control PBS pellet implantation (far right column). ( A-P ) SteREO Lumar microscopy images of GFP-expressing lymphatic vessels (green), mCherry-expressing blood vessels (red) and overlays (right two columns): ( A-D ) prior to implantation, ( E-H ) 3 days post-implantation, ( I-L ) 7 days post-implantation, and ( MP ) 10 days post-implantation. ( Q-T ) Confocal imaging of the same corneas on day 10. Scale bar: 500 μm in ( A-P ) and 200 μm in ( Q-T ). Arrows ( A ): regularly spaced lymphatic vessels penetrating the cornea in the uninjured eye; arrowheads ( E ): new lymphatic vessels budding from the cornea 3 days after VEGF pellet implantation; asterisk ( I ): one potential new lymphatic vessel budding from the cornea on day 7 after the initial phase; circle ( T ): outline of the implanted PBS control pellet.

    Techniques Used: In Vivo, Microscopy, Expressing, Imaging

    27) Product Images from "CXCL1 is critical for pre-metastatic niche formation and metastasis in colorectal cancer"

    Article Title: CXCL1 is critical for pre-metastatic niche formation and metastasis in colorectal cancer

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-16-3199

    VEGF is required for induction of CXCL1, infiltration of MDSC in pre-metastatic liver, and promotion of liver metastasis ( A ) The protein levels of human VEGF-A (left panel) and CXCL1 (right panel) in the cell culture supernatants of indicated cells. ( B ) The protein levels of human VEGF-A, human CXCL1, and mouse CXCL1 in cecal tumors of NSG mice injected with HCT-116/vector or HCT-116/shVEGF-A (left panel), as well as LS-174T/vector or LS-174T/shVEGF-A cells (right panel) without developing liver metastasis. ( C ) The protein levels of mouse CXCL1 in pre-metastatic livers of NSG mice injected with indicated cells as described in panel B. ( D ) The numbers of gMDSCs in pre-metastatic livers of NSG mice injected with indicated cells as described in panel B. ( E – F ) The average numbers of liver metastatic tumors at different size and total that includes all sizes in NSG mice injected with indicated cells as described in panel B. The error bar indicates ± SEM. *p
    Figure Legend Snippet: VEGF is required for induction of CXCL1, infiltration of MDSC in pre-metastatic liver, and promotion of liver metastasis ( A ) The protein levels of human VEGF-A (left panel) and CXCL1 (right panel) in the cell culture supernatants of indicated cells. ( B ) The protein levels of human VEGF-A, human CXCL1, and mouse CXCL1 in cecal tumors of NSG mice injected with HCT-116/vector or HCT-116/shVEGF-A (left panel), as well as LS-174T/vector or LS-174T/shVEGF-A cells (right panel) without developing liver metastasis. ( C ) The protein levels of mouse CXCL1 in pre-metastatic livers of NSG mice injected with indicated cells as described in panel B. ( D ) The numbers of gMDSCs in pre-metastatic livers of NSG mice injected with indicated cells as described in panel B. ( E – F ) The average numbers of liver metastatic tumors at different size and total that includes all sizes in NSG mice injected with indicated cells as described in panel B. The error bar indicates ± SEM. *p

    Techniques Used: Cell Culture, Mouse Assay, Injection, Plasmid Preparation

    A novel mechanism underlying the contribution of primary tumor to pre-metastatic niche formation and liver metastasis Primary malignant cell-secreted VEGF-A stimulates primary tumor-associated macrophages to produce CXCL1 that recruits CXCR2-positive MDSCs from circulatory system into pre-metastatic liver. MDSCs in pre-metastatic liver promote liver metastases via induction of cancer cell survival in a mouse orthotopic model of CRC.
    Figure Legend Snippet: A novel mechanism underlying the contribution of primary tumor to pre-metastatic niche formation and liver metastasis Primary malignant cell-secreted VEGF-A stimulates primary tumor-associated macrophages to produce CXCL1 that recruits CXCR2-positive MDSCs from circulatory system into pre-metastatic liver. MDSCs in pre-metastatic liver promote liver metastases via induction of cancer cell survival in a mouse orthotopic model of CRC.

    Techniques Used:

    Tumor cell-secreted VEGF stimulates tumor-associated macrophages to produce CXCL1 ( A – B ) The percentage (left panes) and numbers (right panel) of DCs, gMDSCs, macrophages (Mϕ), and neutrophils (Neu) in normal cecum of NSG mice injected with HCoEpiC cells and cecal tumors of NSG mice without developing metastasis after injection of HCT-116 cells ( A ) or LS-174T cells ( B ). ( C ) Left panel, data represents the percentage of CXCL1 + macrophages, CXCL1 + gMDSCs, and CXCL1 + DCs in total macrophages, gMDSCs, and DCs in normal cecum and cecal tumors of NSG mice as described in panel A. Right panel, the numbers of CXCL1 + macrophages in normal cecum and cecal tumors of NSG mice as described in panel A. ( D ) Left panel, data represents the percentage of CXCL1 + macrophages, CXCL1 + gMDSCs, and CXCL1 + DCs in total macrophages, gMDSCs, and DCs in normal cecum and cecal tumors of NSG mice as described in panel B. Right panel, the numbers of CXCL1 + macrophages in normal cecum and cecal tumors of NSG mice as described in panel B. ( E – F ) Data represents the percentage of CXCL1 + macrophages in total macrophages (left) and numbers of CXCL1 + macrophages (right panel) in cecal tumors of NSG mice without developing metastasis after injection of HCT-116/vector or HCT-116/shVEGF-A cells ( E ) as well as LS-174T/vector or LS-174T/shVEGF-A cells ( F ). ( G ) Protein levels of mouse CXCL1 in the supernatants of mouse bone marrow-derived macrophages (BMDMs) treated with indicated dose of recombinant human VEGF-A. The error bar indicates ± SEM. *p
    Figure Legend Snippet: Tumor cell-secreted VEGF stimulates tumor-associated macrophages to produce CXCL1 ( A – B ) The percentage (left panes) and numbers (right panel) of DCs, gMDSCs, macrophages (Mϕ), and neutrophils (Neu) in normal cecum of NSG mice injected with HCoEpiC cells and cecal tumors of NSG mice without developing metastasis after injection of HCT-116 cells ( A ) or LS-174T cells ( B ). ( C ) Left panel, data represents the percentage of CXCL1 + macrophages, CXCL1 + gMDSCs, and CXCL1 + DCs in total macrophages, gMDSCs, and DCs in normal cecum and cecal tumors of NSG mice as described in panel A. Right panel, the numbers of CXCL1 + macrophages in normal cecum and cecal tumors of NSG mice as described in panel A. ( D ) Left panel, data represents the percentage of CXCL1 + macrophages, CXCL1 + gMDSCs, and CXCL1 + DCs in total macrophages, gMDSCs, and DCs in normal cecum and cecal tumors of NSG mice as described in panel B. Right panel, the numbers of CXCL1 + macrophages in normal cecum and cecal tumors of NSG mice as described in panel B. ( E – F ) Data represents the percentage of CXCL1 + macrophages in total macrophages (left) and numbers of CXCL1 + macrophages (right panel) in cecal tumors of NSG mice without developing metastasis after injection of HCT-116/vector or HCT-116/shVEGF-A cells ( E ) as well as LS-174T/vector or LS-174T/shVEGF-A cells ( F ). ( G ) Protein levels of mouse CXCL1 in the supernatants of mouse bone marrow-derived macrophages (BMDMs) treated with indicated dose of recombinant human VEGF-A. The error bar indicates ± SEM. *p

    Techniques Used: Mouse Assay, Injection, Plasmid Preparation, Derivative Assay, Recombinant

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    Article Snippet: This tetramethyl-rhodamine (TMR) labeled VEGF (VEGF165 a-TMR) has been used in conjunction with VEGFR2 genetically fused to NanoLuc to provide detailed quantitative information on ligand binding characteristics, the effect of receptor tyrosine kinase inhibitors and the internalization of agonist-receptor complexes in living cells. .. 2.1 Materials All recombinant human VEGF ligand isoforms were purchased from R & D Systems (Abingdon, UK). .. Cediranib, pazopanib, sorafenib and vandetanib were supplied by Sequoia Research Products (Pangbourne, UK).

    Article Title: Inhibition of VEGF and Angiopoietin-2 to Reduce Brain Metastases of Breast Cancer Burden
    Article Snippet: .. Cells were incubated with DMEM (supplemented with 1% FBS) containing various concentrations of Ang-2 (recombinant human Angiopoietin-2, R & D Systems), VEGF (recombinant human VEGF, R & D Systems), L1-10 (Amgen), and/or bevacizumab (AvastinTM Genentech) at 37°C. ..

    Article Title: Cytomegalovirus Impairs Cytotrophoblast-Induced Lymphangiogenesis and Vascular Remodeling in an in Vivo Human Placentation Model
    Article Snippet: Human lymphatic endothelial cells (HLECs) isolated from lymph nodes (ScienCell Research Laboratories, Carlsbad, CA) were plated on fibronectin-coated (1 mg/mL), 96-well plates (5 × 103 cells per well) in endothelial cell medium (ECM) supplemented with endothelial cell growth supplement and 5% FBS (ScienCell Research Laboratories), referred to as growth factor medium (GFM), for 3 days, and then starved for 24 hours in ECM containing 0.5% FBS (control media). .. Media were then replaced with 100 μL of control media, with or without recombinant human VEGF-A, VEGF-C, or bFGF proteins (R & D Systems). .. ECM supplemented with endothelial cell growth supplement and 5% FBS (GFM) was used as a positive control.

    Article Title: CXCL1 is critical for pre-metastatic niche formation and metastasis in colorectal cancer
    Article Snippet: For bone marrow-derived macrophages (BMDMs), bone marrow cells were flushed aseptically from the femurs of NSG mice and cultured in Falcon™ petri dishes (BD Biosciences, Cockeysville, MD) with DMEM medium supplemented with 10% FBS and 10 ng/ml of macrophage colony stimulating factor (M-CSF) for 6 days. .. After BMDMs were cultured in serum-free DMEM medium for 48 hr, the cells were treated with indicated dose of recombinant human VEGF-A (R & D system, Minneapolis, MN) for 24 hr. ..

    Article Title: Comparative Phosphoproteomics Analysis of VEGF and Angiopoietin-1 Signaling Reveals ZO-1 as a Critical Regulator of Endothelial Cell Proliferation *
    Article Snippet: Bovine aortic endothelial cells (BAECs), obtained from VEC Technologies (Rensselaer, NY), were cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (HyClone), 2 m m l -glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. .. BAECs were treated with the recombinant human VEGF-A and recombinant human Ang-1 obtained from R & D System. .. The primary antibodies used were: Anti-phospho-p44/42 MAPK (Thr202/Tyr204) (monoclonal antibody [mAb]), p42/44 MAPK (polyclonal antibody [pAb]), phospho-Ser1179-eNOS (pAb), eNOS (mAb), beta-actin (mAb), phospho-Ser252 p120-catenin (pAb), and BrdU (mAb) from Cell Signaling Technology, Danvers, MA.

    Article Title: Simultaneous in vivo imaging of blood and lymphatic vessel growth in Prox1-GFP/Flk1::myr-mCherry mice
    Article Snippet: .. Pellets contained 10% (w/v) sulfcralfate (Sigma-Aldrich, St. Louis, MO), 12% (w/v) poly-hydroxyethylmethacrylate (HEMA; Sigma-Aldrich) dissolved in absolute ethanol, and recombinant human VEGF-A (R & D Systems, Minneapolis, MN), bFGF, or PBS. ..

    Positive Control:

    Article Title: Neurogenic potential of engineered mesenchymal stem cells overexpressing VEGF
    Article Snippet: Transwells (3.0 μm pore diameter, Corning) seeded with 20,000 control or VEGF-MSCs 24 hours prior were placed over the DRGs in a 24-well plate. .. As a positive control, DRGs were cultured in basal media supplemented with 5 or 100 ng/mL human recombinant VEGF (R & D systems). ..

    Cell Culture:

    Article Title: Neurogenic potential of engineered mesenchymal stem cells overexpressing VEGF
    Article Snippet: Transwells (3.0 μm pore diameter, Corning) seeded with 20,000 control or VEGF-MSCs 24 hours prior were placed over the DRGs in a 24-well plate. .. As a positive control, DRGs were cultured in basal media supplemented with 5 or 100 ng/mL human recombinant VEGF (R & D systems). ..

    Article Title: CXCL1 is critical for pre-metastatic niche formation and metastasis in colorectal cancer
    Article Snippet: For bone marrow-derived macrophages (BMDMs), bone marrow cells were flushed aseptically from the femurs of NSG mice and cultured in Falcon™ petri dishes (BD Biosciences, Cockeysville, MD) with DMEM medium supplemented with 10% FBS and 10 ng/ml of macrophage colony stimulating factor (M-CSF) for 6 days. .. After BMDMs were cultured in serum-free DMEM medium for 48 hr, the cells were treated with indicated dose of recombinant human VEGF-A (R & D system, Minneapolis, MN) for 24 hr. ..

    Incubation:

    Article Title: Inhibition of VEGF and Angiopoietin-2 to Reduce Brain Metastases of Breast Cancer Burden
    Article Snippet: .. Cells were incubated with DMEM (supplemented with 1% FBS) containing various concentrations of Ang-2 (recombinant human Angiopoietin-2, R & D Systems), VEGF (recombinant human VEGF, R & D Systems), L1-10 (Amgen), and/or bevacizumab (AvastinTM Genentech) at 37°C. ..

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    R&D Systems anti vegf bevacizumab similar antibody
    Blockade of both <t>VEGF/VEGFR2</t> and Dll4/Notch1 signaling pathways by HD105 bispecific antibody leads to inhibition of each signaling-induced cellular response. The HD105 bispecific antibody inhibited both the VEGF/VEGFR2 and the Dll4/Notch1 signaling pathways in HUVECs (A). The VEGF/VEGFR2 signaling pathway was monitored by the activation of VEGFR2 and ERK (phosphorylation). The Dll4/Notch1 signaling pathway was monitored by the generation of NICD (Notch-induced intracellular domain). HUVEC sprouting assays were performed in a fibrin gel in the presence of PBS (B), anti-VEGF <t>(bevacizumab-similar)</t> antibody (C), anti-Dll4 antibody (D), or HD105 bispecific antibody (E). Representative images show sprouting tip cells of HUVECs from the beads under basal media (B, arrowheads) and more sprouting under anti-Dll4 antibody treatment (D, arrows) but much less sprouting under anti-VEGF antibody (C) or HD105 bispecific antibody treatment (E). Scale bar (B-E), 150 μm. The bar graph (F) shows the measurement of sprouting HUVECs at 225 μm from beads (n = 20 beads/group, mean ± SE). *, P
    Anti Vegf Bevacizumab Similar Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems human vegf
    Presence of the scAb GLAF-2 and <t>VEGF</t> in tumors of LIVP 6.1.1- or GLV-5b451- injected DT09/06 xenograft mice. ( A ) Western blot analysis of DT09/06 tumor xenografts injected with LIVP 6.1.1 or GLV-5b451 virus (n = 3). The presence of GLAF-2 proteins was performed as described before. Each sample represents an equivalent of 2 mg tumor mass. ( B ) Levels of functional VEGF in tumor lysates determined by <t>ELISA.</t> The graph was plotted using the mean values of each group of three independent measurements. The data are presented as mean values +/− SD. An unpaired t-test was performed revealing significant differences (****P
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    R&D Systems vegf a 165
    MMP-1-PAR1 stimulates secretion of CXCR1/2 chemokines from ovarian carcinoma cells. A , OVCAR-4 cells were stimulated with either 1 nM MMP-1 or PBS buffer in RPMI with 0.1% BSA and conditioned media (CM) was collected after 18 h. The angiogenesis array membranes were incubated with CM from the OVCAR-4 cells and fold change of the spot intensities was quantified using NIH ImageJ software. B ) in OVCAR-4, IGROV-1, OVCAR-3 and OVCAR-4/PAR1 shRNAi ovarian carcinoma cell lines. C–E, ELISA analysis was used to measure IL-8 (C), GRO-α (D), and <t>VEGF-A-165</t> (E) levels in CM from various ovarian carcinoma cell lines that were stimulated with 1 nM MMP-1 in the presence or absence of the PAR1 antagonist pepducin P1pal-7 (3 μM), the small molecule PAR1 antagonist RWJ-56110 (5 μM), or buffer control as indicated. Data (mean ± SE) are from 2–4 experiments performed in duplicate.
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    R&D Systems vegf165
    Human ADSC viability and VEGF expression after transplantation to ischemic murine muscle. A . Representative image of m. tibialis anterior section from “GFP-ADSC” group obtained at day 7 after ischemia induction and GFP-ADSC injection, 50× magnification. GFP-positive cells are distributed in tissue around injection site. B. Analysis of <t>VEGF165</t> content by ELISA in explants culture medium from “ADSC”, “GFP-ADSC”, “VEGF-ADSC” groups obtained at days 3 and 20 after cell trasplantation.
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    Blockade of both VEGF/VEGFR2 and Dll4/Notch1 signaling pathways by HD105 bispecific antibody leads to inhibition of each signaling-induced cellular response. The HD105 bispecific antibody inhibited both the VEGF/VEGFR2 and the Dll4/Notch1 signaling pathways in HUVECs (A). The VEGF/VEGFR2 signaling pathway was monitored by the activation of VEGFR2 and ERK (phosphorylation). The Dll4/Notch1 signaling pathway was monitored by the generation of NICD (Notch-induced intracellular domain). HUVEC sprouting assays were performed in a fibrin gel in the presence of PBS (B), anti-VEGF (bevacizumab-similar) antibody (C), anti-Dll4 antibody (D), or HD105 bispecific antibody (E). Representative images show sprouting tip cells of HUVECs from the beads under basal media (B, arrowheads) and more sprouting under anti-Dll4 antibody treatment (D, arrows) but much less sprouting under anti-VEGF antibody (C) or HD105 bispecific antibody treatment (E). Scale bar (B-E), 150 μm. The bar graph (F) shows the measurement of sprouting HUVECs at 225 μm from beads (n = 20 beads/group, mean ± SE). *, P

    Journal: mAbs

    Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

    doi: 10.1080/19420862.2016.1171432

    Figure Lengend Snippet: Blockade of both VEGF/VEGFR2 and Dll4/Notch1 signaling pathways by HD105 bispecific antibody leads to inhibition of each signaling-induced cellular response. The HD105 bispecific antibody inhibited both the VEGF/VEGFR2 and the Dll4/Notch1 signaling pathways in HUVECs (A). The VEGF/VEGFR2 signaling pathway was monitored by the activation of VEGFR2 and ERK (phosphorylation). The Dll4/Notch1 signaling pathway was monitored by the generation of NICD (Notch-induced intracellular domain). HUVEC sprouting assays were performed in a fibrin gel in the presence of PBS (B), anti-VEGF (bevacizumab-similar) antibody (C), anti-Dll4 antibody (D), or HD105 bispecific antibody (E). Representative images show sprouting tip cells of HUVECs from the beads under basal media (B, arrowheads) and more sprouting under anti-Dll4 antibody treatment (D, arrows) but much less sprouting under anti-VEGF antibody (C) or HD105 bispecific antibody treatment (E). Scale bar (B-E), 150 μm. The bar graph (F) shows the measurement of sprouting HUVECs at 225 μm from beads (n = 20 beads/group, mean ± SE). *, P

    Article Snippet: Increasing concentrations of anti-VEGF (bevacizumab-similar) antibody or HD105 bispecific antibody were mixed with equal volumes of His-tagged recombinant human VEGFR2/Fc (1.65 µg/ml, R & D Systems).

    Techniques: Inhibition, Activation Assay

    Suppression of tumor angiogenesis in cancer xenograft models by HD105 bispecific antibody. Fluorescence micrographs compare the vasculature of A549 human lung cancer tissues in xenograft mice after treatment with PBS (A), anti-VEGF (bevacizumab-similar) antibody (B), anti-mouse Dll4 antibody (C), or mouse HD105 bispecific antibody (D). Scale bar (A-D), 50 μm. The tumor vasculature was stained for CD31 immunoreactivity (green), and the vascular basement was stained for type IV collagen (red). Tumor vessels were decreased after treatment with anti-VEGF (bevacizumab-similar) antibody or mouse HD105 bispecific antibody, whereas tumor vessels were markedly increased after treatment with anti-mouse Dll4 antibody compared to PBS. Higher-resolution images compare the phenotype changes of tumor vessels in detail after PBS (E), anti-VEGF (bevacizumab-similar) antibody (F), anti-mouse Dll4 antibody (G), or mouse HD105 bispecific antibody treatment (H). Scale bar (E-H), 20 μm. The tumor vasculature was stained for CD31 immunoreactivity (red), and the perivascular pericyte was stained for NG2 (green). The nuclei of the tumor tissues were stained by DAPI (4′,6-diamidino-2-phenylindole). Tumor vessels after treatment with anti-mouse Dll4 antibody were conspicuously thinner and more branched than the tumor vessels of other groups. Bar graph (I) measuring tumor vessel density of A549 tumor tissues in xenograft mice confirms the conspicuous increase of tumor vessels after anti-mouse Dll4 antibody treatment but decreases after anti-VEGF (bevacizumab-similar) antibody, mouse HD105 bispecific antibody, or combination treatment with anti-mouse Dll4 antibody and anti-VEGF (bevacizumab-similar) antibody. †, P

    Journal: mAbs

    Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

    doi: 10.1080/19420862.2016.1171432

    Figure Lengend Snippet: Suppression of tumor angiogenesis in cancer xenograft models by HD105 bispecific antibody. Fluorescence micrographs compare the vasculature of A549 human lung cancer tissues in xenograft mice after treatment with PBS (A), anti-VEGF (bevacizumab-similar) antibody (B), anti-mouse Dll4 antibody (C), or mouse HD105 bispecific antibody (D). Scale bar (A-D), 50 μm. The tumor vasculature was stained for CD31 immunoreactivity (green), and the vascular basement was stained for type IV collagen (red). Tumor vessels were decreased after treatment with anti-VEGF (bevacizumab-similar) antibody or mouse HD105 bispecific antibody, whereas tumor vessels were markedly increased after treatment with anti-mouse Dll4 antibody compared to PBS. Higher-resolution images compare the phenotype changes of tumor vessels in detail after PBS (E), anti-VEGF (bevacizumab-similar) antibody (F), anti-mouse Dll4 antibody (G), or mouse HD105 bispecific antibody treatment (H). Scale bar (E-H), 20 μm. The tumor vasculature was stained for CD31 immunoreactivity (red), and the perivascular pericyte was stained for NG2 (green). The nuclei of the tumor tissues were stained by DAPI (4′,6-diamidino-2-phenylindole). Tumor vessels after treatment with anti-mouse Dll4 antibody were conspicuously thinner and more branched than the tumor vessels of other groups. Bar graph (I) measuring tumor vessel density of A549 tumor tissues in xenograft mice confirms the conspicuous increase of tumor vessels after anti-mouse Dll4 antibody treatment but decreases after anti-VEGF (bevacizumab-similar) antibody, mouse HD105 bispecific antibody, or combination treatment with anti-mouse Dll4 antibody and anti-VEGF (bevacizumab-similar) antibody. †, P

    Article Snippet: Increasing concentrations of anti-VEGF (bevacizumab-similar) antibody or HD105 bispecific antibody were mixed with equal volumes of His-tagged recombinant human VEGFR2/Fc (1.65 µg/ml, R & D Systems).

    Techniques: Fluorescence, Mouse Assay, Staining

    Simultaneous binding to VEGF and Dll4 by HD105 bispecific antibody leads to effective blockade of VEGF/VEGFR2 and Dll4/Notch1 interactions. The HD105 bispecific antibody was constructed of the C-terminal of the anti-VEGF (bevacizumab-similar) IgG backbone linked with a single-chain Fv targeting Dll4 (A). The binding affinity of the HD105 bispecific antibody against human VEGF or human Dll4 was determined by Biacore assays (B) and ELISAs (C, D). The K D values of each antibody against VEGF or Dll4 are summarized in Table (B). The HD105 bispecific antibody (closed circle) dose-dependently bound to human VEGF (C) or Dll4 (D). In addition, the HD105 bispecific antibody simultaneously bound to each antigen, human VEGF and human Dll4, in dual-antigen capture ELISAs (E). The anti-Dll4 antibody (open circle in C) or the anti-VEGF (bevacizumab-similar) antibody (open circle in D, E) was used as negative control. Competitive ELISAs demonstrated that the HD105 bispecific antibody inhibited the interaction between VEGF/VEGFR2 (F) or Dll4/Notch1 (G) in a dose-dependent manner. The EC 50 (half maximal effective concentration) values of the anti-VEGF (bevacizumab-similar) antibody (open circle) and HD105 bispecific antibody (closed circle) for VEGF/VEGFR2 inhibition were 2.98 ± 0.5 nM and 2.84 ± 0.41 nM, respectively (F). The EC 50 values of the anti-Dll4 antibody (open circle) and HD105 bispecific antibody (closed circle) were 0.65 ± 0.06 nM and 1.14 ± 0.06 nM, respectively (G).

    Journal: mAbs

    Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

    doi: 10.1080/19420862.2016.1171432

    Figure Lengend Snippet: Simultaneous binding to VEGF and Dll4 by HD105 bispecific antibody leads to effective blockade of VEGF/VEGFR2 and Dll4/Notch1 interactions. The HD105 bispecific antibody was constructed of the C-terminal of the anti-VEGF (bevacizumab-similar) IgG backbone linked with a single-chain Fv targeting Dll4 (A). The binding affinity of the HD105 bispecific antibody against human VEGF or human Dll4 was determined by Biacore assays (B) and ELISAs (C, D). The K D values of each antibody against VEGF or Dll4 are summarized in Table (B). The HD105 bispecific antibody (closed circle) dose-dependently bound to human VEGF (C) or Dll4 (D). In addition, the HD105 bispecific antibody simultaneously bound to each antigen, human VEGF and human Dll4, in dual-antigen capture ELISAs (E). The anti-Dll4 antibody (open circle in C) or the anti-VEGF (bevacizumab-similar) antibody (open circle in D, E) was used as negative control. Competitive ELISAs demonstrated that the HD105 bispecific antibody inhibited the interaction between VEGF/VEGFR2 (F) or Dll4/Notch1 (G) in a dose-dependent manner. The EC 50 (half maximal effective concentration) values of the anti-VEGF (bevacizumab-similar) antibody (open circle) and HD105 bispecific antibody (closed circle) for VEGF/VEGFR2 inhibition were 2.98 ± 0.5 nM and 2.84 ± 0.41 nM, respectively (F). The EC 50 values of the anti-Dll4 antibody (open circle) and HD105 bispecific antibody (closed circle) were 0.65 ± 0.06 nM and 1.14 ± 0.06 nM, respectively (G).

    Article Snippet: Increasing concentrations of anti-VEGF (bevacizumab-similar) antibody or HD105 bispecific antibody were mixed with equal volumes of His-tagged recombinant human VEGFR2/Fc (1.65 µg/ml, R & D Systems).

    Techniques: Binding Assay, Construct, Negative Control, Concentration Assay, Inhibition

    Increase in apoptotic tumor cells in cancer xenograft models treated with HD105 bispecific antibody. Fluorescence micrographs show apoptotic cells stained for activated caspase-3 antibody (red) in SCH human gastric cancer tissues in xenograft mice after treatment with PBS (A), anti-VEGF (bevacizumab-similar) antibody (B), anti-mouse Dll4 antibody (C), and mouse HD105 bispecific antibody (D and E). Scale bar (A-D), 50 μm; (E), 20 μm. Nuclei of the tumor tissues were stained by DAPI (4′,6-diamidino-2-phenylindole, blue). The higher-resolution image confirms that activated caspase-3 antibody was stained in the cytoplasm of the apoptotic cells after mouse HD105 bispecific antibody treatment (E). The bar graph (F) measuring the cell density of apoptotic cells in SCH cancer tissues confirms the significant increase in apoptotic cells after mouse HD105 bispecific antibody treatment. *, P

    Journal: mAbs

    Article Title: Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis

    doi: 10.1080/19420862.2016.1171432

    Figure Lengend Snippet: Increase in apoptotic tumor cells in cancer xenograft models treated with HD105 bispecific antibody. Fluorescence micrographs show apoptotic cells stained for activated caspase-3 antibody (red) in SCH human gastric cancer tissues in xenograft mice after treatment with PBS (A), anti-VEGF (bevacizumab-similar) antibody (B), anti-mouse Dll4 antibody (C), and mouse HD105 bispecific antibody (D and E). Scale bar (A-D), 50 μm; (E), 20 μm. Nuclei of the tumor tissues were stained by DAPI (4′,6-diamidino-2-phenylindole, blue). The higher-resolution image confirms that activated caspase-3 antibody was stained in the cytoplasm of the apoptotic cells after mouse HD105 bispecific antibody treatment (E). The bar graph (F) measuring the cell density of apoptotic cells in SCH cancer tissues confirms the significant increase in apoptotic cells after mouse HD105 bispecific antibody treatment. *, P

    Article Snippet: Increasing concentrations of anti-VEGF (bevacizumab-similar) antibody or HD105 bispecific antibody were mixed with equal volumes of His-tagged recombinant human VEGFR2/Fc (1.65 µg/ml, R & D Systems).

    Techniques: Fluorescence, Staining, Mouse Assay

    Presence of the scAb GLAF-2 and VEGF in tumors of LIVP 6.1.1- or GLV-5b451- injected DT09/06 xenograft mice. ( A ) Western blot analysis of DT09/06 tumor xenografts injected with LIVP 6.1.1 or GLV-5b451 virus (n = 3). The presence of GLAF-2 proteins was performed as described before. Each sample represents an equivalent of 2 mg tumor mass. ( B ) Levels of functional VEGF in tumor lysates determined by ELISA. The graph was plotted using the mean values of each group of three independent measurements. The data are presented as mean values +/− SD. An unpaired t-test was performed revealing significant differences (****P

    Journal: PLoS ONE

    Article Title: Evaluation of a New Recombinant Oncolytic Vaccinia Virus Strain GLV-5b451 for Feline Mammary Carcinoma Therapy

    doi: 10.1371/journal.pone.0104337

    Figure Lengend Snippet: Presence of the scAb GLAF-2 and VEGF in tumors of LIVP 6.1.1- or GLV-5b451- injected DT09/06 xenograft mice. ( A ) Western blot analysis of DT09/06 tumor xenografts injected with LIVP 6.1.1 or GLV-5b451 virus (n = 3). The presence of GLAF-2 proteins was performed as described before. Each sample represents an equivalent of 2 mg tumor mass. ( B ) Levels of functional VEGF in tumor lysates determined by ELISA. The graph was plotted using the mean values of each group of three independent measurements. The data are presented as mean values +/− SD. An unpaired t-test was performed revealing significant differences (****P

    Article Snippet: Concentrations of VEGF were determined by VEGF ELISA kit (Thermo Scientific, Rockford, USA) developed for detection of human VEGF (cross-reacts approximately 82% to recombinant feline VEGF; R & D Systems, Inc., catalog number DVE00, page 11, www.RnDSystem.com ), in accordance with the manufacturer's directions.

    Techniques: Injection, Mouse Assay, Western Blot, Functional Assay, Enzyme-linked Immunosorbent Assay

    Interactions of purified GLAF-2 antibody with feline, murine, and human VEGFs. Affinity and cross reactivity of GLAF-2 was demonstrated by ELISA. Equal concentrations of feline, murine, or human VEGF (100 ng/well) were coated on ELISA plates. Seven two-fold dilutions of purified GLAF-2 protein ranging from 2000 ng/ml to 31.3 ng/ml were incubated with feline, murine and human VEGFs. PBS was used as negative control. For further ELISA experimental conditions see material and methods. ODs obtained for various conc. of GLAF-2 against feline, murine and human VEGF were plotted. ELISA was repeated in three independent experiments. Each value represents the mean (n = 3) +/− standard deviations (SD).

    Journal: PLoS ONE

    Article Title: Evaluation of a New Recombinant Oncolytic Vaccinia Virus Strain GLV-5b451 for Feline Mammary Carcinoma Therapy

    doi: 10.1371/journal.pone.0104337

    Figure Lengend Snippet: Interactions of purified GLAF-2 antibody with feline, murine, and human VEGFs. Affinity and cross reactivity of GLAF-2 was demonstrated by ELISA. Equal concentrations of feline, murine, or human VEGF (100 ng/well) were coated on ELISA plates. Seven two-fold dilutions of purified GLAF-2 protein ranging from 2000 ng/ml to 31.3 ng/ml were incubated with feline, murine and human VEGFs. PBS was used as negative control. For further ELISA experimental conditions see material and methods. ODs obtained for various conc. of GLAF-2 against feline, murine and human VEGF were plotted. ELISA was repeated in three independent experiments. Each value represents the mean (n = 3) +/− standard deviations (SD).

    Article Snippet: Concentrations of VEGF were determined by VEGF ELISA kit (Thermo Scientific, Rockford, USA) developed for detection of human VEGF (cross-reacts approximately 82% to recombinant feline VEGF; R & D Systems, Inc., catalog number DVE00, page 11, www.RnDSystem.com ), in accordance with the manufacturer's directions.

    Techniques: Purification, Enzyme-linked Immunosorbent Assay, Incubation, Negative Control

    MMP-1-PAR1 stimulates secretion of CXCR1/2 chemokines from ovarian carcinoma cells. A , OVCAR-4 cells were stimulated with either 1 nM MMP-1 or PBS buffer in RPMI with 0.1% BSA and conditioned media (CM) was collected after 18 h. The angiogenesis array membranes were incubated with CM from the OVCAR-4 cells and fold change of the spot intensities was quantified using NIH ImageJ software. B ) in OVCAR-4, IGROV-1, OVCAR-3 and OVCAR-4/PAR1 shRNAi ovarian carcinoma cell lines. C–E, ELISA analysis was used to measure IL-8 (C), GRO-α (D), and VEGF-A-165 (E) levels in CM from various ovarian carcinoma cell lines that were stimulated with 1 nM MMP-1 in the presence or absence of the PAR1 antagonist pepducin P1pal-7 (3 μM), the small molecule PAR1 antagonist RWJ-56110 (5 μM), or buffer control as indicated. Data (mean ± SE) are from 2–4 experiments performed in duplicate.

    Journal: Cancer research

    Article Title: Identification of a metalloprotease-chemokine signaling system in the ovarian cancer microenvironment: implications for anti-angiogenic therapy

    doi: 10.1158/0008-5472.CAN-09-4341

    Figure Lengend Snippet: MMP-1-PAR1 stimulates secretion of CXCR1/2 chemokines from ovarian carcinoma cells. A , OVCAR-4 cells were stimulated with either 1 nM MMP-1 or PBS buffer in RPMI with 0.1% BSA and conditioned media (CM) was collected after 18 h. The angiogenesis array membranes were incubated with CM from the OVCAR-4 cells and fold change of the spot intensities was quantified using NIH ImageJ software. B ) in OVCAR-4, IGROV-1, OVCAR-3 and OVCAR-4/PAR1 shRNAi ovarian carcinoma cell lines. C–E, ELISA analysis was used to measure IL-8 (C), GRO-α (D), and VEGF-A-165 (E) levels in CM from various ovarian carcinoma cell lines that were stimulated with 1 nM MMP-1 in the presence or absence of the PAR1 antagonist pepducin P1pal-7 (3 μM), the small molecule PAR1 antagonist RWJ-56110 (5 μM), or buffer control as indicated. Data (mean ± SE) are from 2–4 experiments performed in duplicate.

    Article Snippet: Quantikine ELISAs for human IL-8, GRO-α and VEGF-A-165, were obtained from R & D Systems and used as recommended by the manufacturer.

    Techniques: Incubation, Software, Enzyme-linked Immunosorbent Assay

    Human ADSC viability and VEGF expression after transplantation to ischemic murine muscle. A . Representative image of m. tibialis anterior section from “GFP-ADSC” group obtained at day 7 after ischemia induction and GFP-ADSC injection, 50× magnification. GFP-positive cells are distributed in tissue around injection site. B. Analysis of VEGF165 content by ELISA in explants culture medium from “ADSC”, “GFP-ADSC”, “VEGF-ADSC” groups obtained at days 3 and 20 after cell trasplantation.

    Journal: Journal of Translational Medicine

    Article Title: Transplantation of modified human adipose derived stromal cells expressing VEGF165 results in more efficient angiogenic response in ischemic skeletal muscle

    doi: 10.1186/1479-5876-11-138

    Figure Lengend Snippet: Human ADSC viability and VEGF expression after transplantation to ischemic murine muscle. A . Representative image of m. tibialis anterior section from “GFP-ADSC” group obtained at day 7 after ischemia induction and GFP-ADSC injection, 50× magnification. GFP-positive cells are distributed in tissue around injection site. B. Analysis of VEGF165 content by ELISA in explants culture medium from “ADSC”, “GFP-ADSC”, “VEGF-ADSC” groups obtained at days 3 and 20 after cell trasplantation.

    Article Snippet: VEGF165 concentration in condition media samples was measured using human VEGF Quantikine Kit (R & D Systems, USA) following manufacturer’s protocol.

    Techniques: Expressing, Transplantation Assay, Injection, Enzyme-linked Immunosorbent Assay

    Validation of VEGF165 expression in AAV-modified VEGF-ADSC. A. VEGFA expression level in human ADSC 10 days after AAV transduction determined by quantitative PCR. B, C. Analysis of VEGF secretion by GFP-ADSC, VEGF-ADSC and unmodified cells using ELISA ( B ) and immunoblotting ( C ). In immunosorbent assay protein content was determined in conditioned media samples obtained at days 7 and 30 post genetic modification of ADSC.

    Journal: Journal of Translational Medicine

    Article Title: Transplantation of modified human adipose derived stromal cells expressing VEGF165 results in more efficient angiogenic response in ischemic skeletal muscle

    doi: 10.1186/1479-5876-11-138

    Figure Lengend Snippet: Validation of VEGF165 expression in AAV-modified VEGF-ADSC. A. VEGFA expression level in human ADSC 10 days after AAV transduction determined by quantitative PCR. B, C. Analysis of VEGF secretion by GFP-ADSC, VEGF-ADSC and unmodified cells using ELISA ( B ) and immunoblotting ( C ). In immunosorbent assay protein content was determined in conditioned media samples obtained at days 7 and 30 post genetic modification of ADSC.

    Article Snippet: VEGF165 concentration in condition media samples was measured using human VEGF Quantikine Kit (R & D Systems, USA) following manufacturer’s protocol.

    Techniques: Expressing, Modification, Transduction, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay