vegfr 1  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Name:
    FLT1 VEGFR1 Recombinant Human Protein without Catalytic Activity Domain
    Description:
    Fms related tyrosine kinase 1 FLT1 VEGFR1 recombinant human protein is supplied as a lyophilized powder It is suitable for use in analysis of protein structure In general recombinant proteins can also be used as an immunogen as a protein standard or in cell biology research applications Since this product is a truncated protein and does not contain intracellular catalytic activity domain it is not suitable for use in enzyme activity studies This recombinant protein was expressed from a DNA sequence encoding the extracellular domain Met 1 Asn 756 of human FLT1 P17948 1 fused to a polyhistidine tag at the C terminus Activity Measured by its ability to inhibit the VEGF dependent proliferation of human umbilical vein endothelial cells HUVEC Conn G et al 1990 Proc Natl Acad Sci USA 87 1323 The ED50 for this effect is typically 5 30 ng ml in the presence of 5 ng ml human VEGF165 Formulation Lyophilized in PBS pH7 4 5 Mannitol 5 Trehalose and 0 02 Tween 80 Reconstitution Dissolve the protein in sterile double distilled water to a concentration of 0 2 mg ml or lower It is recommended that the protein be aliquoted and be used as soon as possible Store aliquots under sterile conditions at 20°C Avoid repeated freeze thaw cycles Expiration Date Expires one year from date of receipt when stored as instructed This protein is manufactured by SINO Biological
    Catalog Number:
    10136h08h250
    Price:
    None
    Applications:
    Enzyme & Protein Activity Assays|Protein Assay Controls, Reference Standards & Accessories|Protein Assays and Analysis|Protein Biology
    Category:
    Proteins Enzymes Peptides
    Buy from Supplier


    Structured Review

    Thermo Fisher vegfr 1
    Influence of <t>VEGFR‐1</t> silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Fms related tyrosine kinase 1 FLT1 VEGFR1 recombinant human protein is supplied as a lyophilized powder It is suitable for use in analysis of protein structure In general recombinant proteins can also be used as an immunogen as a protein standard or in cell biology research applications Since this product is a truncated protein and does not contain intracellular catalytic activity domain it is not suitable for use in enzyme activity studies This recombinant protein was expressed from a DNA sequence encoding the extracellular domain Met 1 Asn 756 of human FLT1 P17948 1 fused to a polyhistidine tag at the C terminus Activity Measured by its ability to inhibit the VEGF dependent proliferation of human umbilical vein endothelial cells HUVEC Conn G et al 1990 Proc Natl Acad Sci USA 87 1323 The ED50 for this effect is typically 5 30 ng ml in the presence of 5 ng ml human VEGF165 Formulation Lyophilized in PBS pH7 4 5 Mannitol 5 Trehalose and 0 02 Tween 80 Reconstitution Dissolve the protein in sterile double distilled water to a concentration of 0 2 mg ml or lower It is recommended that the protein be aliquoted and be used as soon as possible Store aliquots under sterile conditions at 20°C Avoid repeated freeze thaw cycles Expiration Date Expires one year from date of receipt when stored as instructed This protein is manufactured by SINO Biological
    https://www.bioz.com/result/vegfr 1/product/Thermo Fisher
    Average 97 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    vegfr 1 - by Bioz Stars, 2020-09
    97/100 stars

    Images

    1) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    2) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    3) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    4) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    5) Product Images from "Vascular Endothelial Growth Factor Isoforms and Their Receptors Are Expressed in Human Osteoarthritic Cartilage"

    Article Title: Vascular Endothelial Growth Factor Isoforms and Their Receptors Are Expressed in Human Osteoarthritic Cartilage

    Journal: The American Journal of Pathology

    doi:

    RT-PCR for the expression of VEGF isoforms and their receptors (VEGFR-1, VEGFR-2, and NRP-1) in OA and NOR cartilage. Total RNA was extracted from OA and NOR cartilage and reverse-transcribed into cDNA followed by a PCR reaction using specific primer pairs for VEGF and VEGF receptors as described in Materials and Methods.
    Figure Legend Snippet: RT-PCR for the expression of VEGF isoforms and their receptors (VEGFR-1, VEGFR-2, and NRP-1) in OA and NOR cartilage. Total RNA was extracted from OA and NOR cartilage and reverse-transcribed into cDNA followed by a PCR reaction using specific primer pairs for VEGF and VEGF receptors as described in Materials and Methods.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction

    Immunolocalization of VEGFR-1 and VEGFR-2 in NOR and OA cartilage. Immunostaining of VEGFR-1 ( A and C ) and VEGFR-2 ( B and D ) was performed as described in Materials and Methods. A and B: NOR cartilage, Mankin grade 0. C and D: OA cartilage, Mankin grade 8. Note that OA chondrocytes in the transitional and radial zones are positively immunostained for VEGFR-1 ( C , arrows ) and VEGFR-2 ( D , arrows ). Scale bar, 100 μm.
    Figure Legend Snippet: Immunolocalization of VEGFR-1 and VEGFR-2 in NOR and OA cartilage. Immunostaining of VEGFR-1 ( A and C ) and VEGFR-2 ( B and D ) was performed as described in Materials and Methods. A and B: NOR cartilage, Mankin grade 0. C and D: OA cartilage, Mankin grade 8. Note that OA chondrocytes in the transitional and radial zones are positively immunostained for VEGFR-1 ( C , arrows ) and VEGFR-2 ( D , arrows ). Scale bar, 100 μm.

    Techniques Used: Immunostaining

    Immunohistochemistry of VEGFR-1, VEGFR-2, CD31, and von Willebrand factor in cultured OA chondrocytes. OA chondrocytes ( A–E ) and human umbilical vein endothelial cells ( F ) were cultured on chamber slides and immunostained with antibodies against VEGFR-1 ( A ), VEGFR-2 ( B ), CD31 ( C ), and von Willebrand factor ( D and F ) or nonimmune mouse IgG ( E ) as described in Materials and Methods. Note that OA chondrocytes are positively stained with the antibodies to VEGFR-1 ( A ) and VEGFR-2 ( B ), but negative for CD31 ( C ) and von Willebrand factor ( D ). Human umbilical vein endothelial cells (a positive control) are stained with the antibody to von Willebrand factor ( F ). Scale bar, 25 μm.
    Figure Legend Snippet: Immunohistochemistry of VEGFR-1, VEGFR-2, CD31, and von Willebrand factor in cultured OA chondrocytes. OA chondrocytes ( A–E ) and human umbilical vein endothelial cells ( F ) were cultured on chamber slides and immunostained with antibodies against VEGFR-1 ( A ), VEGFR-2 ( B ), CD31 ( C ), and von Willebrand factor ( D and F ) or nonimmune mouse IgG ( E ) as described in Materials and Methods. Note that OA chondrocytes are positively stained with the antibodies to VEGFR-1 ( A ) and VEGFR-2 ( B ), but negative for CD31 ( C ) and von Willebrand factor ( D ). Human umbilical vein endothelial cells (a positive control) are stained with the antibody to von Willebrand factor ( F ). Scale bar, 25 μm.

    Techniques Used: Immunohistochemistry, Cell Culture, Staining, Positive Control

    6) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    7) Product Images from "Notch1 Deficiency Results in Decreased Inflammation during Wound Healing and Regulates Vascular Endothelial Growth Factor Receptor-1 and Inflammatory Cytokine Expression in Macrophages"

    Article Title: Notch1 Deficiency Results in Decreased Inflammation during Wound Healing and Regulates Vascular Endothelial Growth Factor Receptor-1 and Inflammatory Cytokine Expression in Macrophages

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1000720

    Decreased Notch signaling and VEGFR-1 expression in circulating monocytes from Notch1 +/− mice. A, Notch signaling was assessed in peripheral blood leukocytes from Notch reporter mice harboring a CSL-responsive GFP transgene using flow cytometry to detect GFP expression. B , Circulating monocytes from Notch1 +/− mice expressed decreased VEGFR-1 compared with WT littermates ( left ), whereas expression of CD11b was unchanged ( right ). WT, wild-type.
    Figure Legend Snippet: Decreased Notch signaling and VEGFR-1 expression in circulating monocytes from Notch1 +/− mice. A, Notch signaling was assessed in peripheral blood leukocytes from Notch reporter mice harboring a CSL-responsive GFP transgene using flow cytometry to detect GFP expression. B , Circulating monocytes from Notch1 +/− mice expressed decreased VEGFR-1 compared with WT littermates ( left ), whereas expression of CD11b was unchanged ( right ). WT, wild-type.

    Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Cytometry

    Notch signaling is induced in BMM in response to LPS/IFN-γ and IL-4. BMM were stimulated with 100 ng/ml LPS and 100 U/ml IFN-γ or 5 ng/ml IL-4 overnight .Transcripts were assessed by quantitative RT-PCR. A, iNOS ( left ) and arginase (right) expression in response to LPS/IFN-γ or IL-4 stimulation. B , Stimulation with LPS/IFN-γ induced expression of Notch1 and Jagged1. C, Stimulation with IL-4 induced expression of Jagged1, but not Notch1. D, Both IL-4 ( left ) and LPS/IFN-γ ( right ) induced expression of the Notch target gene Hey1. E , Both IL-4 ( left ) and LPS/IFN-γ ( right ) induced expression VEGFR-1. Quantitative RT-PCR results represent reactions performed in triplicate and normalized to expression of P0 (6SD). Data are representative of at least three independent experiments. *p
    Figure Legend Snippet: Notch signaling is induced in BMM in response to LPS/IFN-γ and IL-4. BMM were stimulated with 100 ng/ml LPS and 100 U/ml IFN-γ or 5 ng/ml IL-4 overnight .Transcripts were assessed by quantitative RT-PCR. A, iNOS ( left ) and arginase (right) expression in response to LPS/IFN-γ or IL-4 stimulation. B , Stimulation with LPS/IFN-γ induced expression of Notch1 and Jagged1. C, Stimulation with IL-4 induced expression of Jagged1, but not Notch1. D, Both IL-4 ( left ) and LPS/IFN-γ ( right ) induced expression of the Notch target gene Hey1. E , Both IL-4 ( left ) and LPS/IFN-γ ( right ) induced expression VEGFR-1. Quantitative RT-PCR results represent reactions performed in triplicate and normalized to expression of P0 (6SD). Data are representative of at least three independent experiments. *p

    Techniques Used: Quantitative RT-PCR, Expressing

    Notch mediates VEGFR-1 induction in response to LPS/IFN-γ, but not IL-4. A, Flow cytometry of BMM after overnight stimulation showed increased surface expression of VEGFR-1 in response to LPS/IFN-γ ( left ), but not IL-4 ( right ). Coincubation of BMM with 400 nM GSI inhibited induction of transcript ( B ) and protein ( C ) levels of VEGFR-1 after overnight stimulation with LPS/IFN-γ. D , Incubation with Jag1Fc-conditioned medium inhibited induction of VEGFR-1 in response to LPS. E , Induction of transcript levels of VEGFR-1 in response to IL-4 was unaffected by GSI. F , Surface expression of VEGFR-1 was unaffected by IL-4, but coincubation with GSI led to a slight decrease in VEGFR-1 expression. G , Immunoblotting for arginase expression in whole-cell protein extracts from IL-4–stimulated BMM, with or without GSI ( upper panel ). The same membrane was probed for α-tubulin as a loading control (lower panel). Quantitative RT-PCR results represent reactions performed in triplicate and normalized to expression of P0 (±SD). Data are representative of at least three independent experiments. * p
    Figure Legend Snippet: Notch mediates VEGFR-1 induction in response to LPS/IFN-γ, but not IL-4. A, Flow cytometry of BMM after overnight stimulation showed increased surface expression of VEGFR-1 in response to LPS/IFN-γ ( left ), but not IL-4 ( right ). Coincubation of BMM with 400 nM GSI inhibited induction of transcript ( B ) and protein ( C ) levels of VEGFR-1 after overnight stimulation with LPS/IFN-γ. D , Incubation with Jag1Fc-conditioned medium inhibited induction of VEGFR-1 in response to LPS. E , Induction of transcript levels of VEGFR-1 in response to IL-4 was unaffected by GSI. F , Surface expression of VEGFR-1 was unaffected by IL-4, but coincubation with GSI led to a slight decrease in VEGFR-1 expression. G , Immunoblotting for arginase expression in whole-cell protein extracts from IL-4–stimulated BMM, with or without GSI ( upper panel ). The same membrane was probed for α-tubulin as a loading control (lower panel). Quantitative RT-PCR results represent reactions performed in triplicate and normalized to expression of P0 (±SD). Data are representative of at least three independent experiments. * p

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Incubation, Quantitative RT-PCR

    Notch1, not Notch4, regulates baseline levels of VEGFR-1 in BMM. A, A lentiviral construct encoding the constitutively active N1IC was transduced into BMM and transcripts were assessed by quantitative RT-PCR 48 h postinfection. N1IC trans-duction led to increased VEGFR-1 expression ( left ), as well as increased expression of the Notch target gene Hey1 ( right ). B , Incubation of resting BMM with GSI overnight led to decreased transcript ( left ) and protein ( right ) levels of VEGFR-1. C , BMM from Notch mutant mice were analyzed for VEGFR-1 expression by flow cytometry. Notch1 +/− BMM had decreased surface expression of VEGFR-1 ( left ), whereas VEGFR-1 expression in Notch4 −/− BMM was largely unchanged ( right ) compared with WT BMM. D, Quantitative RT-PCR demonstrated that BMM from Notch4 −/− mice induce VEGFR-1 in response to LPS/IFN-γ to a similar degree as do WT BMM, whereas loss of Notch1 leads to diminished induction of VEGFR-1. E , Production of NO in culture supernatant from WT or Notch1 +/− BMM was assessed by Griess reagent. Cells were stimulated overnight with LPS/IFN-γ. Some cells were coincubated with GSI. Quantitative RT-PCR results represent reactions performed in triplicate and normalized to expression of P0 (±SD). Data are representative of at least three independent experiments. * p
    Figure Legend Snippet: Notch1, not Notch4, regulates baseline levels of VEGFR-1 in BMM. A, A lentiviral construct encoding the constitutively active N1IC was transduced into BMM and transcripts were assessed by quantitative RT-PCR 48 h postinfection. N1IC trans-duction led to increased VEGFR-1 expression ( left ), as well as increased expression of the Notch target gene Hey1 ( right ). B , Incubation of resting BMM with GSI overnight led to decreased transcript ( left ) and protein ( right ) levels of VEGFR-1. C , BMM from Notch mutant mice were analyzed for VEGFR-1 expression by flow cytometry. Notch1 +/− BMM had decreased surface expression of VEGFR-1 ( left ), whereas VEGFR-1 expression in Notch4 −/− BMM was largely unchanged ( right ) compared with WT BMM. D, Quantitative RT-PCR demonstrated that BMM from Notch4 −/− mice induce VEGFR-1 in response to LPS/IFN-γ to a similar degree as do WT BMM, whereas loss of Notch1 leads to diminished induction of VEGFR-1. E , Production of NO in culture supernatant from WT or Notch1 +/− BMM was assessed by Griess reagent. Cells were stimulated overnight with LPS/IFN-γ. Some cells were coincubated with GSI. Quantitative RT-PCR results represent reactions performed in triplicate and normalized to expression of P0 (±SD). Data are representative of at least three independent experiments. * p

    Techniques Used: Construct, Quantitative RT-PCR, Expressing, Incubation, Mutagenesis, Mouse Assay, Flow Cytometry, Cytometry

    8) Product Images from "Leptin-induced transphosphorylation of vascular endothelial growth factor receptor increases Notch and stimulates endothelial cell angiogenic transformation"

    Article Title: Leptin-induced transphosphorylation of vascular endothelial growth factor receptor increases Notch and stimulates endothelial cell angiogenic transformation

    Journal: The international journal of biochemistry & cell biology

    doi: 10.1016/j.biocel.2016.08.023

    A functional OB-R/VEGFR/Notch axis stimulates leptin-induced angiogenic features in endothelial cells Leptin-induces Notch expression in endothelial cells through activation of VEGFR-1 and VEGFR-2 in the absence of VEGF. Leptin induces VEGFR-2 expression and transphosphorylation at Y951, Y966, and Y1175, that in turn induces the expression of Notch genes/proteins. These effects of leptin increase endothelial cell proliferation, S-phase progression, and tube formation. Inhibition of OB-R, VEGFR-2 or Notch signaling negatively impacts leptin pro-angiogenic effects in endothelial cells.
    Figure Legend Snippet: A functional OB-R/VEGFR/Notch axis stimulates leptin-induced angiogenic features in endothelial cells Leptin-induces Notch expression in endothelial cells through activation of VEGFR-1 and VEGFR-2 in the absence of VEGF. Leptin induces VEGFR-2 expression and transphosphorylation at Y951, Y966, and Y1175, that in turn induces the expression of Notch genes/proteins. These effects of leptin increase endothelial cell proliferation, S-phase progression, and tube formation. Inhibition of OB-R, VEGFR-2 or Notch signaling negatively impacts leptin pro-angiogenic effects in endothelial cells.

    Techniques Used: Functional Assay, Expressing, Activation Assay, Inhibition

    Inhibition of kinase activity and knockdown of VEGF receptors decrease leptin induction of Notch expression in endothelial cells A. Leptin induction of Notch receptors in HUVEC requires VEGFR2 kinase activity . Western blot representative results from the reduction of leptin-induced Notch receptors via VEGFR kinase inhibition. HUVEC were treated for 24 h with leptin (1.2 nM), VEGFR-2 inhibitor SU5416 (5 µmol/l) and positive control VEGF (25 ng/ml). B. Leptin induction of Notch proteins in HUVEC requires VEGFR-2 gene activity . Represented here are western blot results from the reduction of leptin-induced Notch via VEGFR-2 SiRNA knockdown. Results show the effects of VEGFR-2 SiRNA (1 µg) after 6 h on leptin-induced expression of Notch receptors and ligands in HUVEC. C. Leptin induction of Notch proteins in HUVEC is reduced by knockdown of VEGFR-1 and VEGFR-2 gene activities . Western blot representative results from the reduction of leptin-induced Notch via VEGFR-1 and VEGFR-2 SiRNA knockdown. D. Leptin induction of Notch mRNA in HUVEC is reduced by knockdown of VEGFR-1 and VEGFR-2 gene activities . Quantitative results from real-time PCR of Notch, VEGFR-1 and VEGFR-2 mRNA expression in HUVEC treated with leptin and VEGFR-1 and VEGFR-2 SiRNA. Beta-actin or GAPDH were used as loading controls. Relative protein expression was calculated as percentage to basal. Histograms show densitometric analysis of Notch protein expression using NIH image J software. RNA expression was calculated by normalizing values to GAPDH mRNA. Relative mRNA expression was calculated to basal. Data is presented as an average ± s.d. from three independent experiments. Su: SU5416; SiControl: siRNA control. a: p
    Figure Legend Snippet: Inhibition of kinase activity and knockdown of VEGF receptors decrease leptin induction of Notch expression in endothelial cells A. Leptin induction of Notch receptors in HUVEC requires VEGFR2 kinase activity . Western blot representative results from the reduction of leptin-induced Notch receptors via VEGFR kinase inhibition. HUVEC were treated for 24 h with leptin (1.2 nM), VEGFR-2 inhibitor SU5416 (5 µmol/l) and positive control VEGF (25 ng/ml). B. Leptin induction of Notch proteins in HUVEC requires VEGFR-2 gene activity . Represented here are western blot results from the reduction of leptin-induced Notch via VEGFR-2 SiRNA knockdown. Results show the effects of VEGFR-2 SiRNA (1 µg) after 6 h on leptin-induced expression of Notch receptors and ligands in HUVEC. C. Leptin induction of Notch proteins in HUVEC is reduced by knockdown of VEGFR-1 and VEGFR-2 gene activities . Western blot representative results from the reduction of leptin-induced Notch via VEGFR-1 and VEGFR-2 SiRNA knockdown. D. Leptin induction of Notch mRNA in HUVEC is reduced by knockdown of VEGFR-1 and VEGFR-2 gene activities . Quantitative results from real-time PCR of Notch, VEGFR-1 and VEGFR-2 mRNA expression in HUVEC treated with leptin and VEGFR-1 and VEGFR-2 SiRNA. Beta-actin or GAPDH were used as loading controls. Relative protein expression was calculated as percentage to basal. Histograms show densitometric analysis of Notch protein expression using NIH image J software. RNA expression was calculated by normalizing values to GAPDH mRNA. Relative mRNA expression was calculated to basal. Data is presented as an average ± s.d. from three independent experiments. Su: SU5416; SiControl: siRNA control. a: p

    Techniques Used: Inhibition, Activity Assay, Expressing, Western Blot, Positive Control, Real-time Polymerase Chain Reaction, Software, RNA Expression

    VEGF receptors are involved in leptin-induced Notch expression in endothelial cells A. VEGFR expression in PAEC . WB representative results of VEGFR in PAEC transfected with VEGFR-1 and VEGFR-2. GAPDH was used as a loading control. B. Leptin receptor (OB-R) expression in PAEC . WB representative results from OB-R immunoprecipitation (IP) in PAEC wild type, and PAEC transfected with VEGFR-1 and VEGFR-2. Cells lysates (25 µg protein) were incubated at 4°C for 16 h with anti-OB-R antibody (0.5 µg). C. Leptin induction of Notch proteins in PAEC-VEGFR-1 . Western blot (WB) representative results of leptin-induced Notch proteins in PAEC transfected with VEGFR-1. D. Leptin dose-dependent induction of Notch proteins in PAEC-VEGFR-2 . PAEC transfected with VEGFR-1 and VEGFR-2 were cultured in medium containing leptin (0.6,1.2, and 6.2 nM) for 24 h. Cell lysates were used to determine Notch protein expression after treatment. GAPDH was used as a loading control. Histograms show densitometric analysis of protein expression normalized to GAPDH as determined using NIH image J software. Relative protein expression was calculated as percentage to basal. WB analysis did not show basal or leptin-induced expression of Notch proteins in wild type PAEC. Protein G-agarose beads were added for IP/WB analysis. Data is presented as an average ± s.d. from three independent experiments. * p
    Figure Legend Snippet: VEGF receptors are involved in leptin-induced Notch expression in endothelial cells A. VEGFR expression in PAEC . WB representative results of VEGFR in PAEC transfected with VEGFR-1 and VEGFR-2. GAPDH was used as a loading control. B. Leptin receptor (OB-R) expression in PAEC . WB representative results from OB-R immunoprecipitation (IP) in PAEC wild type, and PAEC transfected with VEGFR-1 and VEGFR-2. Cells lysates (25 µg protein) were incubated at 4°C for 16 h with anti-OB-R antibody (0.5 µg). C. Leptin induction of Notch proteins in PAEC-VEGFR-1 . Western blot (WB) representative results of leptin-induced Notch proteins in PAEC transfected with VEGFR-1. D. Leptin dose-dependent induction of Notch proteins in PAEC-VEGFR-2 . PAEC transfected with VEGFR-1 and VEGFR-2 were cultured in medium containing leptin (0.6,1.2, and 6.2 nM) for 24 h. Cell lysates were used to determine Notch protein expression after treatment. GAPDH was used as a loading control. Histograms show densitometric analysis of protein expression normalized to GAPDH as determined using NIH image J software. Relative protein expression was calculated as percentage to basal. WB analysis did not show basal or leptin-induced expression of Notch proteins in wild type PAEC. Protein G-agarose beads were added for IP/WB analysis. Data is presented as an average ± s.d. from three independent experiments. * p

    Techniques Used: Expressing, Western Blot, Transfection, Immunoprecipitation, Incubation, Cell Culture, Software

    VEGF receptors are involved in leptin-induced Notch mRNA in endothelial cells A. Leptin dose-dependent induction of Notch mRNA in PAEC wild type; B. PAEC-VEGFR-1 and C. PAEC-VEGFR-2 . Quantitative results from real-time PCR of Notch mRNA expression in PAEC wild type and PAEC transfected with VEGFR-1 and VEGFR-2. Cells were cultured in medium containing leptin (0.6,1.2, and 6.2 nM) for 24 h. Cell lysates were used to determine Notch mRNA expression after treatment. RNA expression was calculated by normalizing values to GAPDH mRNA. Relative mRNA expression was calculated to basal. Data is presented as an average ± s.d. from three independent experiments. * p
    Figure Legend Snippet: VEGF receptors are involved in leptin-induced Notch mRNA in endothelial cells A. Leptin dose-dependent induction of Notch mRNA in PAEC wild type; B. PAEC-VEGFR-1 and C. PAEC-VEGFR-2 . Quantitative results from real-time PCR of Notch mRNA expression in PAEC wild type and PAEC transfected with VEGFR-1 and VEGFR-2. Cells were cultured in medium containing leptin (0.6,1.2, and 6.2 nM) for 24 h. Cell lysates were used to determine Notch mRNA expression after treatment. RNA expression was calculated by normalizing values to GAPDH mRNA. Relative mRNA expression was calculated to basal. Data is presented as an average ± s.d. from three independent experiments. * p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Transfection, Cell Culture, RNA Expression

    9) Product Images from "The anti-vascular endothelial growth factor receptor-1 monoclonal antibody D16F7 inhibits invasiveness of human glioblastoma and glioblastoma stem cells"

    Article Title: The anti-vascular endothelial growth factor receptor-1 monoclonal antibody D16F7 inhibits invasiveness of human glioblastoma and glioblastoma stem cells

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-017-0577-2

    D16F7 inhibitory effects on VEGF-A or PlGF-induced migration of GSCs. a Detection of VEGFR-1 and VEGFR-2 transcripts in GSC lines was performed by qRT-PCR. Results indicate relative mRNA expression and are the mean ± SD of three independent determinations. b VEGFR-1 protein levels were analyzed by Western blotting using β-actin detection as loading control. HUVEC and M14 cells were used as positive and negative controls, respectively. c Migration of GSCs in response to PlGF or VEGF-A in the absence or in the presence of 5 μg/ml D16F7 was analyzed as described in Fig. 1c legend. Photographs from a representative experiment out of three with #213 cells are shown (40× magnification). d Histograms represent the mean ± SD ( n = 3) of migrated cells/microscopic field. Results of statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test were as follows: in #213, PlGF vs NS or PlGF vs PlGF + D16F7, p
    Figure Legend Snippet: D16F7 inhibitory effects on VEGF-A or PlGF-induced migration of GSCs. a Detection of VEGFR-1 and VEGFR-2 transcripts in GSC lines was performed by qRT-PCR. Results indicate relative mRNA expression and are the mean ± SD of three independent determinations. b VEGFR-1 protein levels were analyzed by Western blotting using β-actin detection as loading control. HUVEC and M14 cells were used as positive and negative controls, respectively. c Migration of GSCs in response to PlGF or VEGF-A in the absence or in the presence of 5 μg/ml D16F7 was analyzed as described in Fig. 1c legend. Photographs from a representative experiment out of three with #213 cells are shown (40× magnification). d Histograms represent the mean ± SD ( n = 3) of migrated cells/microscopic field. Results of statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test were as follows: in #213, PlGF vs NS or PlGF vs PlGF + D16F7, p

    Techniques Used: Migration, Quantitative RT-PCR, Expressing, Western Blot

    Inhibition by D16F7 of VEGF-A and PlGF-induced phosphorylation of VEGFR-1 at Tyr 1213 in GBM cells over-expressing VEGFR-1. a VEGFR-1 mRNA levels in U87-derived clones transfected with control (U87-CTR6) or VEGFR-1 expressing (U87-MF1 and U87-MF24) vectors was analyzed by RT-PCR. Amplified products were separated on 1% agarose gels and results are representative of one out of two different experiments giving comparable results. b VEGFR-1 protein levels in U87-derived clones transfected with control or VEGFR-1 expressing vectors were analyzed by Western blotting. Numbers below immunoblot lanes indicate VEGFR-1/β-actin optical density (O.D.) ratios. c Western blotting of total or phosphorylated VEGFR-1 (pVEGFR-1) at tyrosine 1213 and total or phosphorylated Erk1/2 (pErk) in untreated or D16F7 (1 or 10 μg/ml) pre-treated U87-MF24 cells in response to PlGF or VEGF-A. Histograms represent the densitometric quantification of band intensities in the corresponding immunoblots, expressed as pVEGFR-1/VEGFR-1 ratio relative to untreated control, after normalization for β-actin expression. Normalized pVEGFR-1/VEGFR-1 or pErk/Erk protein ratio in untreated cells was considered equal to 1. d Histogram represents the mean ± SD percentage inhibition values of PlGF or VEGF-A-induced VEGFR-1 phosphorylation or Erk1/2 phosphorylation in U87-MF24 cells after treatment with 1 and 10 μg/ml D16F7, calculated from immunoblot densitometric analysis of three independent experiments. e For spheroid invasion assay U87-MF24 cells were embedded in matrigel in the absence or presence of D16F7 (10 μg/ml) and PlGF (50 ng/ml). Representative pictures of spheroids taken at 24, 48 and 72 h after embedding cells in matrigel (40× magnification) are shown; NS, non-stimulated cells. Relative invasion was quantified as described in Fig. 1d legend. Data are expressed as mean ± SD ( n = 6–10) and results of statistical analysis were as follows: PlGF vs NS, PlGF vs D16F7 or PlGF vs PlGF + D16F7, p
    Figure Legend Snippet: Inhibition by D16F7 of VEGF-A and PlGF-induced phosphorylation of VEGFR-1 at Tyr 1213 in GBM cells over-expressing VEGFR-1. a VEGFR-1 mRNA levels in U87-derived clones transfected with control (U87-CTR6) or VEGFR-1 expressing (U87-MF1 and U87-MF24) vectors was analyzed by RT-PCR. Amplified products were separated on 1% agarose gels and results are representative of one out of two different experiments giving comparable results. b VEGFR-1 protein levels in U87-derived clones transfected with control or VEGFR-1 expressing vectors were analyzed by Western blotting. Numbers below immunoblot lanes indicate VEGFR-1/β-actin optical density (O.D.) ratios. c Western blotting of total or phosphorylated VEGFR-1 (pVEGFR-1) at tyrosine 1213 and total or phosphorylated Erk1/2 (pErk) in untreated or D16F7 (1 or 10 μg/ml) pre-treated U87-MF24 cells in response to PlGF or VEGF-A. Histograms represent the densitometric quantification of band intensities in the corresponding immunoblots, expressed as pVEGFR-1/VEGFR-1 ratio relative to untreated control, after normalization for β-actin expression. Normalized pVEGFR-1/VEGFR-1 or pErk/Erk protein ratio in untreated cells was considered equal to 1. d Histogram represents the mean ± SD percentage inhibition values of PlGF or VEGF-A-induced VEGFR-1 phosphorylation or Erk1/2 phosphorylation in U87-MF24 cells after treatment with 1 and 10 μg/ml D16F7, calculated from immunoblot densitometric analysis of three independent experiments. e For spheroid invasion assay U87-MF24 cells were embedded in matrigel in the absence or presence of D16F7 (10 μg/ml) and PlGF (50 ng/ml). Representative pictures of spheroids taken at 24, 48 and 72 h after embedding cells in matrigel (40× magnification) are shown; NS, non-stimulated cells. Relative invasion was quantified as described in Fig. 1d legend. Data are expressed as mean ± SD ( n = 6–10) and results of statistical analysis were as follows: PlGF vs NS, PlGF vs D16F7 or PlGF vs PlGF + D16F7, p

    Techniques Used: Inhibition, Expressing, Derivative Assay, Clone Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Invasion Assay

    D16F7 inhibits ECM invasion of P3, EGFRwt + and EGFRvIII + GSCs that express VEGFR-1. a EGFR or mutated EGFRvIII protein levels were analyzed by Western blotting using β-actin detection as loading control. b Detection of VEGFR-1 and VEGFR-2 transcripts was performed by qRT-PCR. Results indicate relative mRNA expression and are the mean ± SD of three independent determinations. EGFRvIII + vs P3 and EGFRwt + cells, p
    Figure Legend Snippet: D16F7 inhibits ECM invasion of P3, EGFRwt + and EGFRvIII + GSCs that express VEGFR-1. a EGFR or mutated EGFRvIII protein levels were analyzed by Western blotting using β-actin detection as loading control. b Detection of VEGFR-1 and VEGFR-2 transcripts was performed by qRT-PCR. Results indicate relative mRNA expression and are the mean ± SD of three independent determinations. EGFRvIII + vs P3 and EGFRwt + cells, p

    Techniques Used: Western Blot, Quantitative RT-PCR, Expressing

    D16F7 inhibitory effects on VEGF-A or PlGF-induced migration and ECM invasion in human GBM cells expressing VEGFR-1. a Detection of VEGFR-1 and VEGFR-2 transcripts in GBM cell lines was performed by qRT-PCR. Results indicate relative mRNA expression and are the mean ± SD of three independent determinations. b PlGF and VEGF-A secretion was quantified by ELISA (mean ± SD, n = 3). c Migration of U87 cells in response to PlGF or VEGF-A was evaluated in the absence (not treated, NT) or presence of 5 μg/ml D16F7; NS, non-stimulated cells. Representative photographs of U87 cells are shown (100× magnification). Histograms represent the mean ± SD ( n = 3) of migrated cells/microscopic field. Results of statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test were as follows: PlGF vs NS, PlGF vs D16F7 or PlGF vs PlGF + D16F7 and VEGF-A vs NS, VEGF-A vs D16F7 or VEGF-A vs VEGF-A + D16F7, p
    Figure Legend Snippet: D16F7 inhibitory effects on VEGF-A or PlGF-induced migration and ECM invasion in human GBM cells expressing VEGFR-1. a Detection of VEGFR-1 and VEGFR-2 transcripts in GBM cell lines was performed by qRT-PCR. Results indicate relative mRNA expression and are the mean ± SD of three independent determinations. b PlGF and VEGF-A secretion was quantified by ELISA (mean ± SD, n = 3). c Migration of U87 cells in response to PlGF or VEGF-A was evaluated in the absence (not treated, NT) or presence of 5 μg/ml D16F7; NS, non-stimulated cells. Representative photographs of U87 cells are shown (100× magnification). Histograms represent the mean ± SD ( n = 3) of migrated cells/microscopic field. Results of statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test were as follows: PlGF vs NS, PlGF vs D16F7 or PlGF vs PlGF + D16F7 and VEGF-A vs NS, VEGF-A vs D16F7 or VEGF-A vs VEGF-A + D16F7, p

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

    10) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    11) Product Images from "Differential Expression of VEGF, EG-VEGF, and VEGF Receptors in Human Placentas from Normal and Pre-eclamptic Pregnancies"

    Article Title: Differential Expression of VEGF, EG-VEGF, and VEGF Receptors in Human Placentas from Normal and Pre-eclamptic Pregnancies

    Journal: The Journal of Clinical Endocrinology and Metabolism

    doi:

    Immunolocalization of VEGF, VEGFR-1, VEGFR-2, NP-1, and NP-2 in human placentas from normal pregnancy. The tissues sections were incubated either with a rabbit antibody against each indicated antigen, or preimmune rabbit IgG (4 μg/ml; IgG). *, lumen of blood vessels; arrow heads, endothelial cells. All panels are in the same magnification. Bar = 40 μm.
    Figure Legend Snippet: Immunolocalization of VEGF, VEGFR-1, VEGFR-2, NP-1, and NP-2 in human placentas from normal pregnancy. The tissues sections were incubated either with a rabbit antibody against each indicated antigen, or preimmune rabbit IgG (4 μg/ml; IgG). *, lumen of blood vessels; arrow heads, endothelial cells. All panels are in the same magnification. Bar = 40 μm.

    Techniques Used: Incubation

    RT-PCR analysis for VEGF, EG-VEGF, VEGFR-1, VEGFR-2, NP-1, and NP-2 in human placentas. The total RNA samples (0.5 μg/gene) from one normal placenta were used for PCR amplification. The PCR products were confirmed by sequencing and used as standards for the real-time PCR.
    Figure Legend Snippet: RT-PCR analysis for VEGF, EG-VEGF, VEGFR-1, VEGFR-2, NP-1, and NP-2 in human placentas. The total RNA samples (0.5 μg/gene) from one normal placenta were used for PCR amplification. The PCR products were confirmed by sequencing and used as standards for the real-time PCR.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Sequencing, Real-time Polymerase Chain Reaction

    Real-time PCR analysis of the mRNA levels of VEGF, EG-VEGF, VEGFR-1, VEGFR-2, NP-1, and NP-2 in human placentas from normal and PE pregnancies. For each gene, cDNA was amplified from total RNA (2 μg/sample) of normal or PE placentas and 4 μl of cDNA per sample was used for real-time PCR. The mRNA levels were normalized to β-actin. For each sample, the real-time PCR reaction was performed in duplicate or triplicate for each mRNA. Data are expressed as means ± SE. *, differs from its corresponding N ( p
    Figure Legend Snippet: Real-time PCR analysis of the mRNA levels of VEGF, EG-VEGF, VEGFR-1, VEGFR-2, NP-1, and NP-2 in human placentas from normal and PE pregnancies. For each gene, cDNA was amplified from total RNA (2 μg/sample) of normal or PE placentas and 4 μl of cDNA per sample was used for real-time PCR. The mRNA levels were normalized to β-actin. For each sample, the real-time PCR reaction was performed in duplicate or triplicate for each mRNA. Data are expressed as means ± SE. *, differs from its corresponding N ( p

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    Western blot analysis for VEGF, VEGFR-1, VEGFR-2, NP-1, and NP-2 in human placentas from normal and PE pregnancies. (A) A representative Western blot for VEGF, VEGFR-1, VEGFR–2, NP-1, NP–2, and GAPDH. (a) VEGF: proteins were first immunoprecipitated with a rabbit polyclonal antibody against VEGF, followed by immunobloting with a mouse-monoclonal anti-human VEGF antibody. Recombinant human VEGF165 (at 20 kD) and VEGF121 (at 18 kD) were used as positive controls. (b) VEGFR-1 and VEGFR-2: proteins were separated and detected. One major band for each protein was observed at 180 Kd and 235 Kd for VEGFR-1 and VEGFR-2 respectively, corresponding to its positive control HUVEC. The protein levels of VEGFR-1 and VEGFR-2 were normalized to GAPDH. (c) NP-1 and NP-2: proteins were first immunoprecipitated with a rabbit polyclonal NP-1 or NP-2 antibody, followed by immunobloting with a mouse monoclonal anti NP-1 or NP-2 antibody. One major band was located at ~130 Kd, corresponding to their HUVEC positive controls. (B) Protein levels of VEGF and its four receptors. *, differs from its corresponding N, p
    Figure Legend Snippet: Western blot analysis for VEGF, VEGFR-1, VEGFR-2, NP-1, and NP-2 in human placentas from normal and PE pregnancies. (A) A representative Western blot for VEGF, VEGFR-1, VEGFR–2, NP-1, NP–2, and GAPDH. (a) VEGF: proteins were first immunoprecipitated with a rabbit polyclonal antibody against VEGF, followed by immunobloting with a mouse-monoclonal anti-human VEGF antibody. Recombinant human VEGF165 (at 20 kD) and VEGF121 (at 18 kD) were used as positive controls. (b) VEGFR-1 and VEGFR-2: proteins were separated and detected. One major band for each protein was observed at 180 Kd and 235 Kd for VEGFR-1 and VEGFR-2 respectively, corresponding to its positive control HUVEC. The protein levels of VEGFR-1 and VEGFR-2 were normalized to GAPDH. (c) NP-1 and NP-2: proteins were first immunoprecipitated with a rabbit polyclonal NP-1 or NP-2 antibody, followed by immunobloting with a mouse monoclonal anti NP-1 or NP-2 antibody. One major band was located at ~130 Kd, corresponding to their HUVEC positive controls. (B) Protein levels of VEGF and its four receptors. *, differs from its corresponding N, p

    Techniques Used: Western Blot, Immunoprecipitation, Recombinant, Positive Control

    12) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    13) Product Images from "The anti-vascular endothelial growth factor receptor-1 monoclonal antibody D16F7 inhibits invasiveness of human glioblastoma and glioblastoma stem cells"

    Article Title: The anti-vascular endothelial growth factor receptor-1 monoclonal antibody D16F7 inhibits invasiveness of human glioblastoma and glioblastoma stem cells

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-017-0577-2

    D16F7 inhibitory effects on VEGF-A or PlGF-induced migration of GSCs. a Detection of VEGFR-1 and VEGFR-2 transcripts in GSC lines was performed by qRT-PCR. Results indicate relative mRNA expression and are the mean ± SD of three independent determinations. b VEGFR-1 protein levels were analyzed by Western blotting using β-actin detection as loading control. HUVEC and M14 cells were used as positive and negative controls, respectively. c Migration of GSCs in response to PlGF or VEGF-A in the absence or in the presence of 5 μg/ml D16F7 was analyzed as described in Fig. 1c legend. Photographs from a representative experiment out of three with #213 cells are shown (40× magnification). d Histograms represent the mean ± SD ( n = 3) of migrated cells/microscopic field. Results of statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test were as follows: in #213, PlGF vs NS or PlGF vs PlGF + D16F7, p
    Figure Legend Snippet: D16F7 inhibitory effects on VEGF-A or PlGF-induced migration of GSCs. a Detection of VEGFR-1 and VEGFR-2 transcripts in GSC lines was performed by qRT-PCR. Results indicate relative mRNA expression and are the mean ± SD of three independent determinations. b VEGFR-1 protein levels were analyzed by Western blotting using β-actin detection as loading control. HUVEC and M14 cells were used as positive and negative controls, respectively. c Migration of GSCs in response to PlGF or VEGF-A in the absence or in the presence of 5 μg/ml D16F7 was analyzed as described in Fig. 1c legend. Photographs from a representative experiment out of three with #213 cells are shown (40× magnification). d Histograms represent the mean ± SD ( n = 3) of migrated cells/microscopic field. Results of statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test were as follows: in #213, PlGF vs NS or PlGF vs PlGF + D16F7, p

    Techniques Used: Migration, Quantitative RT-PCR, Expressing, Western Blot

    Inhibition by D16F7 of VEGF-A and PlGF-induced phosphorylation of VEGFR-1 at Tyr 1213 in GBM cells over-expressing VEGFR-1. a VEGFR-1 mRNA levels in U87-derived clones transfected with control (U87-CTR6) or VEGFR-1 expressing (U87-MF1 and U87-MF24) vectors was analyzed by RT-PCR. Amplified products were separated on 1% agarose gels and results are representative of one out of two different experiments giving comparable results. b VEGFR-1 protein levels in U87-derived clones transfected with control or VEGFR-1 expressing vectors were analyzed by Western blotting. Numbers below immunoblot lanes indicate VEGFR-1/β-actin optical density (O.D.) ratios. c Western blotting of total or phosphorylated VEGFR-1 (pVEGFR-1) at tyrosine 1213 and total or phosphorylated Erk1/2 (pErk) in untreated or D16F7 (1 or 10 μg/ml) pre-treated U87-MF24 cells in response to PlGF or VEGF-A. Histograms represent the densitometric quantification of band intensities in the corresponding immunoblots, expressed as pVEGFR-1/VEGFR-1 ratio relative to untreated control, after normalization for β-actin expression. Normalized pVEGFR-1/VEGFR-1 or pErk/Erk protein ratio in untreated cells was considered equal to 1. d Histogram represents the mean ± SD percentage inhibition values of PlGF or VEGF-A-induced VEGFR-1 phosphorylation or Erk1/2 phosphorylation in U87-MF24 cells after treatment with 1 and 10 μg/ml D16F7, calculated from immunoblot densitometric analysis of three independent experiments. e For spheroid invasion assay U87-MF24 cells were embedded in matrigel in the absence or presence of D16F7 (10 μg/ml) and PlGF (50 ng/ml). Representative pictures of spheroids taken at 24, 48 and 72 h after embedding cells in matrigel (40× magnification) are shown; NS, non-stimulated cells. Relative invasion was quantified as described in Fig. 1d legend. Data are expressed as mean ± SD ( n = 6–10) and results of statistical analysis were as follows: PlGF vs NS, PlGF vs D16F7 or PlGF vs PlGF + D16F7, p
    Figure Legend Snippet: Inhibition by D16F7 of VEGF-A and PlGF-induced phosphorylation of VEGFR-1 at Tyr 1213 in GBM cells over-expressing VEGFR-1. a VEGFR-1 mRNA levels in U87-derived clones transfected with control (U87-CTR6) or VEGFR-1 expressing (U87-MF1 and U87-MF24) vectors was analyzed by RT-PCR. Amplified products were separated on 1% agarose gels and results are representative of one out of two different experiments giving comparable results. b VEGFR-1 protein levels in U87-derived clones transfected with control or VEGFR-1 expressing vectors were analyzed by Western blotting. Numbers below immunoblot lanes indicate VEGFR-1/β-actin optical density (O.D.) ratios. c Western blotting of total or phosphorylated VEGFR-1 (pVEGFR-1) at tyrosine 1213 and total or phosphorylated Erk1/2 (pErk) in untreated or D16F7 (1 or 10 μg/ml) pre-treated U87-MF24 cells in response to PlGF or VEGF-A. Histograms represent the densitometric quantification of band intensities in the corresponding immunoblots, expressed as pVEGFR-1/VEGFR-1 ratio relative to untreated control, after normalization for β-actin expression. Normalized pVEGFR-1/VEGFR-1 or pErk/Erk protein ratio in untreated cells was considered equal to 1. d Histogram represents the mean ± SD percentage inhibition values of PlGF or VEGF-A-induced VEGFR-1 phosphorylation or Erk1/2 phosphorylation in U87-MF24 cells after treatment with 1 and 10 μg/ml D16F7, calculated from immunoblot densitometric analysis of three independent experiments. e For spheroid invasion assay U87-MF24 cells were embedded in matrigel in the absence or presence of D16F7 (10 μg/ml) and PlGF (50 ng/ml). Representative pictures of spheroids taken at 24, 48 and 72 h after embedding cells in matrigel (40× magnification) are shown; NS, non-stimulated cells. Relative invasion was quantified as described in Fig. 1d legend. Data are expressed as mean ± SD ( n = 6–10) and results of statistical analysis were as follows: PlGF vs NS, PlGF vs D16F7 or PlGF vs PlGF + D16F7, p

    Techniques Used: Inhibition, Expressing, Derivative Assay, Clone Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Invasion Assay

    D16F7 inhibits ECM invasion of P3, EGFRwt + and EGFRvIII + GSCs that express VEGFR-1. a EGFR or mutated EGFRvIII protein levels were analyzed by Western blotting using β-actin detection as loading control. b Detection of VEGFR-1 and VEGFR-2 transcripts was performed by qRT-PCR. Results indicate relative mRNA expression and are the mean ± SD of three independent determinations. EGFRvIII + vs P3 and EGFRwt + cells, p
    Figure Legend Snippet: D16F7 inhibits ECM invasion of P3, EGFRwt + and EGFRvIII + GSCs that express VEGFR-1. a EGFR or mutated EGFRvIII protein levels were analyzed by Western blotting using β-actin detection as loading control. b Detection of VEGFR-1 and VEGFR-2 transcripts was performed by qRT-PCR. Results indicate relative mRNA expression and are the mean ± SD of three independent determinations. EGFRvIII + vs P3 and EGFRwt + cells, p

    Techniques Used: Western Blot, Quantitative RT-PCR, Expressing

    D16F7 inhibitory effects on VEGF-A or PlGF-induced migration and ECM invasion in human GBM cells expressing VEGFR-1. a Detection of VEGFR-1 and VEGFR-2 transcripts in GBM cell lines was performed by qRT-PCR. Results indicate relative mRNA expression and are the mean ± SD of three independent determinations. b PlGF and VEGF-A secretion was quantified by ELISA (mean ± SD, n = 3). c Migration of U87 cells in response to PlGF or VEGF-A was evaluated in the absence (not treated, NT) or presence of 5 μg/ml D16F7; NS, non-stimulated cells. Representative photographs of U87 cells are shown (100× magnification). Histograms represent the mean ± SD ( n = 3) of migrated cells/microscopic field. Results of statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test were as follows: PlGF vs NS, PlGF vs D16F7 or PlGF vs PlGF + D16F7 and VEGF-A vs NS, VEGF-A vs D16F7 or VEGF-A vs VEGF-A + D16F7, p
    Figure Legend Snippet: D16F7 inhibitory effects on VEGF-A or PlGF-induced migration and ECM invasion in human GBM cells expressing VEGFR-1. a Detection of VEGFR-1 and VEGFR-2 transcripts in GBM cell lines was performed by qRT-PCR. Results indicate relative mRNA expression and are the mean ± SD of three independent determinations. b PlGF and VEGF-A secretion was quantified by ELISA (mean ± SD, n = 3). c Migration of U87 cells in response to PlGF or VEGF-A was evaluated in the absence (not treated, NT) or presence of 5 μg/ml D16F7; NS, non-stimulated cells. Representative photographs of U87 cells are shown (100× magnification). Histograms represent the mean ± SD ( n = 3) of migrated cells/microscopic field. Results of statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test were as follows: PlGF vs NS, PlGF vs D16F7 or PlGF vs PlGF + D16F7 and VEGF-A vs NS, VEGF-A vs D16F7 or VEGF-A vs VEGF-A + D16F7, p

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

    14) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    15) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    16) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    17) Product Images from "The Prognostic Value of the Combination of Low VEGFR-1 and High VEGFR-2 Expression in Endothelial Cells of Colorectal Cancer"

    Article Title: The Prognostic Value of the Combination of Low VEGFR-1 and High VEGFR-2 Expression in Endothelial Cells of Colorectal Cancer

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19113536

    Distribution of VEGFR-1 and VEGFR-2 expression in ECs relative to survival: ( A ) LI values ≤ 5% for VEGFR-1 expression in CRC ECs identify the majority of patients with a low metastasis risk. Red/blue dots identify metastatic/metastasis-free patients, respectively. ( B ) LI values ≤ 5% for VEGFR-1 expression in CRC ECs identify the majority of patients with improved overall survival. Red/blue dots identify dead/alive patients respectively. ( C ) LI values ≥ 33% for VEGFR-2 expression in CRC ECs identify the majority of patients with a low metastasis risk. Red/blue dots identify metastatic/metastasis-free patients respectively.
    Figure Legend Snippet: Distribution of VEGFR-1 and VEGFR-2 expression in ECs relative to survival: ( A ) LI values ≤ 5% for VEGFR-1 expression in CRC ECs identify the majority of patients with a low metastasis risk. Red/blue dots identify metastatic/metastasis-free patients, respectively. ( B ) LI values ≤ 5% for VEGFR-1 expression in CRC ECs identify the majority of patients with improved overall survival. Red/blue dots identify dead/alive patients respectively. ( C ) LI values ≥ 33% for VEGFR-2 expression in CRC ECs identify the majority of patients with a low metastasis risk. Red/blue dots identify metastatic/metastasis-free patients respectively.

    Techniques Used: Expressing

    Prognostic value of the combination of the VEGFR-1 and VEGFR-2 expression in ECs of CRC: ( A ) Metastasis-free survival curves of patients dichotomized based on the combination of VEGFR-1-LI and VEGFR-2-LI ( p = 0.002). ( B ) Overall survival curves of patients dichotomized in a similar manner ( p = 0.029). Complete and censured data are shown as dots and crosses, respectively. Solid lines identify patients with ECs characterized by a VEGFR-1 LI ≤ 5% and a VEGFR-2 LI > 33%. Dotted lines identify patients with ECs characterized by a VEGFR-1 LI > 5% and/or a VEGFR-2 LI ≤ 33%.
    Figure Legend Snippet: Prognostic value of the combination of the VEGFR-1 and VEGFR-2 expression in ECs of CRC: ( A ) Metastasis-free survival curves of patients dichotomized based on the combination of VEGFR-1-LI and VEGFR-2-LI ( p = 0.002). ( B ) Overall survival curves of patients dichotomized in a similar manner ( p = 0.029). Complete and censured data are shown as dots and crosses, respectively. Solid lines identify patients with ECs characterized by a VEGFR-1 LI ≤ 5% and a VEGFR-2 LI > 33%. Dotted lines identify patients with ECs characterized by a VEGFR-1 LI > 5% and/or a VEGFR-2 LI ≤ 33%.

    Techniques Used: Expressing

    Heterogeneous VEGF, VEGFR-1 and VEGFR-2 expression in colorectal cancer (CRC) endothelial cells (ECs): VEGF expression in ECs was heterogeneous with an labelling index (LI) range from 10.9 to 90% ( A , D ); Anti-VEGFR-1 immunostaining shows rare positivity with an LI range from nearly 0 ( B ) to 20% ( E ) in endothelial cells. VEGFR-2 shows a much wider immunostaining, with an LI range from 10 ( C ) to 72% ( F ). (Magnification: 400×). The arrows are pointed at vessels.
    Figure Legend Snippet: Heterogeneous VEGF, VEGFR-1 and VEGFR-2 expression in colorectal cancer (CRC) endothelial cells (ECs): VEGF expression in ECs was heterogeneous with an labelling index (LI) range from 10.9 to 90% ( A , D ); Anti-VEGFR-1 immunostaining shows rare positivity with an LI range from nearly 0 ( B ) to 20% ( E ) in endothelial cells. VEGFR-2 shows a much wider immunostaining, with an LI range from 10 ( C ) to 72% ( F ). (Magnification: 400×). The arrows are pointed at vessels.

    Techniques Used: Expressing, Immunostaining

    Quantitative image analysis on Tissue MicroArray (TMA): ( A ) After digitization of the TMA slides with a NanoZoomer Digital Slide Scanner (Hamamatsu, Hamamatsu City, Japan), blood vessels, highlighted by anti-CD34 immunostaining, were manually marked on the digital vascular endothelial growth factor receptor (VEGFR) slides by a pathologist using the NDP.view software. The tissue core diameter is 600 µm. ( B ) The delineated regions were imported into the Visiopharm software to quantify the VEGF, VEGFR-1 or VEGFR-2 expression levels using the labelling index (LI), i.e., the percentage of the immunoreactive area within the vessel area.
    Figure Legend Snippet: Quantitative image analysis on Tissue MicroArray (TMA): ( A ) After digitization of the TMA slides with a NanoZoomer Digital Slide Scanner (Hamamatsu, Hamamatsu City, Japan), blood vessels, highlighted by anti-CD34 immunostaining, were manually marked on the digital vascular endothelial growth factor receptor (VEGFR) slides by a pathologist using the NDP.view software. The tissue core diameter is 600 µm. ( B ) The delineated regions were imported into the Visiopharm software to quantify the VEGF, VEGFR-1 or VEGFR-2 expression levels using the labelling index (LI), i.e., the percentage of the immunoreactive area within the vessel area.

    Techniques Used: Microarray, Immunostaining, Software, Expressing

    Prognostic value of VEGFR-1 and VEGFR-2 expression in EC of CRC: ( A ) Metastasis-free survival curves of patients dichotomized based on VEGFR-1 LI ≤ (solid line) or > (dotted line) 5% ( p = 0.063). ( B ) Overall survival curves of patients dichotomized based on VEGFR-1 LI ≤ (solid line) or > (dotted line) 5% ( p = 0.013). ( C ) Metastasis-free survival curves of patients dichotomized based on VEGFR-2 LI ≤ (solid line) or > (dotted line) 33% ( p = 0.002). Complete and censured data are shown as dots and crosses, respectively.
    Figure Legend Snippet: Prognostic value of VEGFR-1 and VEGFR-2 expression in EC of CRC: ( A ) Metastasis-free survival curves of patients dichotomized based on VEGFR-1 LI ≤ (solid line) or > (dotted line) 5% ( p = 0.063). ( B ) Overall survival curves of patients dichotomized based on VEGFR-1 LI ≤ (solid line) or > (dotted line) 5% ( p = 0.013). ( C ) Metastasis-free survival curves of patients dichotomized based on VEGFR-2 LI ≤ (solid line) or > (dotted line) 33% ( p = 0.002). Complete and censured data are shown as dots and crosses, respectively.

    Techniques Used: Expressing

    18) Product Images from "Durable Responses with the Metronomic Regimen RT-PEPC in Elderly Patients with Recurrent Mantle Cell Lymphoma"

    Article Title: Durable Responses with the Metronomic Regimen RT-PEPC in Elderly Patients with Recurrent Mantle Cell Lymphoma

    Journal: Cancer

    doi: 10.1002/cncr.25055

    Angiogenesis and lymphangiogenesis characterization in MCL A. VEGFR-1 is expressed by MCL B-cells ( A1 ) and neovessels (v = vessel) ( A2 ) by immunofluorescence analysis, and appears to be overexpressed in neoplastic B-cells (n=5) compared to normal B-cells (n=5) and MCL cell line Jeko-1 by quantitative PCR analysis ( A3 ). B. VEGFR-2 expression in neovasculature. B1: VEGFR-2 immunohistochemistry on frozen tissue section. B2: A majority of VEGFR-2 + tumor endothelial cells coexpress CD34 by immunofluorescence analysis (white arrows). B3: VEGFR-2 vessels (V) are situated in proximity to VEGF-producing tumor cells ( B3 ). C. Lymphangiogenesis as marked by VEGFR-3 + ( C1 ), podoplanin + ( C2 ) and Lyve-1 + ( C3 ). Some lymphatic vessels appear to co-express CD34. Black arrows point to CD34 + podoplanin + vessels, and asterisks point to CD34 + lyve-1 + vessels.
    Figure Legend Snippet: Angiogenesis and lymphangiogenesis characterization in MCL A. VEGFR-1 is expressed by MCL B-cells ( A1 ) and neovessels (v = vessel) ( A2 ) by immunofluorescence analysis, and appears to be overexpressed in neoplastic B-cells (n=5) compared to normal B-cells (n=5) and MCL cell line Jeko-1 by quantitative PCR analysis ( A3 ). B. VEGFR-2 expression in neovasculature. B1: VEGFR-2 immunohistochemistry on frozen tissue section. B2: A majority of VEGFR-2 + tumor endothelial cells coexpress CD34 by immunofluorescence analysis (white arrows). B3: VEGFR-2 vessels (V) are situated in proximity to VEGF-producing tumor cells ( B3 ). C. Lymphangiogenesis as marked by VEGFR-3 + ( C1 ), podoplanin + ( C2 ) and Lyve-1 + ( C3 ). Some lymphatic vessels appear to co-express CD34. Black arrows point to CD34 + podoplanin + vessels, and asterisks point to CD34 + lyve-1 + vessels.

    Techniques Used: Immunofluorescence, Real-time Polymerase Chain Reaction, Expressing, Immunohistochemistry

    19) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    20) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    21) Product Images from "The Prognostic Value of the Combination of Low VEGFR-1 and High VEGFR-2 Expression in Endothelial Cells of Colorectal Cancer"

    Article Title: The Prognostic Value of the Combination of Low VEGFR-1 and High VEGFR-2 Expression in Endothelial Cells of Colorectal Cancer

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19113536

    Distribution of VEGFR-1 and VEGFR-2 expression in ECs relative to survival: ( A ) LI values ≤ 5% for VEGFR-1 expression in CRC ECs identify the majority of patients with a low metastasis risk. Red/blue dots identify metastatic/metastasis-free patients, respectively. ( B ) LI values ≤ 5% for VEGFR-1 expression in CRC ECs identify the majority of patients with improved overall survival. Red/blue dots identify dead/alive patients respectively. ( C ) LI values ≥ 33% for VEGFR-2 expression in CRC ECs identify the majority of patients with a low metastasis risk. Red/blue dots identify metastatic/metastasis-free patients respectively.
    Figure Legend Snippet: Distribution of VEGFR-1 and VEGFR-2 expression in ECs relative to survival: ( A ) LI values ≤ 5% for VEGFR-1 expression in CRC ECs identify the majority of patients with a low metastasis risk. Red/blue dots identify metastatic/metastasis-free patients, respectively. ( B ) LI values ≤ 5% for VEGFR-1 expression in CRC ECs identify the majority of patients with improved overall survival. Red/blue dots identify dead/alive patients respectively. ( C ) LI values ≥ 33% for VEGFR-2 expression in CRC ECs identify the majority of patients with a low metastasis risk. Red/blue dots identify metastatic/metastasis-free patients respectively.

    Techniques Used: Expressing

    Heterogeneous VEGF, VEGFR-1 and VEGFR-2 expression in colorectal cancer (CRC) endothelial cells (ECs): VEGF expression in ECs was heterogeneous with an labelling index (LI) range from 10.9 to 90% ( A , D ); Anti-VEGFR-1 immunostaining shows rare positivity with an LI range from nearly 0 ( B ) to 20% ( E ) in endothelial cells. VEGFR-2 shows a much wider immunostaining, with an LI range from 10 ( C ) to 72% ( F ). (Magnification: 400×). The arrows are pointed at vessels.
    Figure Legend Snippet: Heterogeneous VEGF, VEGFR-1 and VEGFR-2 expression in colorectal cancer (CRC) endothelial cells (ECs): VEGF expression in ECs was heterogeneous with an labelling index (LI) range from 10.9 to 90% ( A , D ); Anti-VEGFR-1 immunostaining shows rare positivity with an LI range from nearly 0 ( B ) to 20% ( E ) in endothelial cells. VEGFR-2 shows a much wider immunostaining, with an LI range from 10 ( C ) to 72% ( F ). (Magnification: 400×). The arrows are pointed at vessels.

    Techniques Used: Expressing, Immunostaining

    Quantitative image analysis on Tissue MicroArray (TMA): ( A ) After digitization of the TMA slides with a NanoZoomer Digital Slide Scanner (Hamamatsu, Hamamatsu City, Japan), blood vessels, highlighted by anti-CD34 immunostaining, were manually marked on the digital vascular endothelial growth factor receptor (VEGFR) slides by a pathologist using the NDP.view software. The tissue core diameter is 600 µm. ( B ) The delineated regions were imported into the Visiopharm software to quantify the VEGF, VEGFR-1 or VEGFR-2 expression levels using the labelling index (LI), i.e., the percentage of the immunoreactive area within the vessel area.
    Figure Legend Snippet: Quantitative image analysis on Tissue MicroArray (TMA): ( A ) After digitization of the TMA slides with a NanoZoomer Digital Slide Scanner (Hamamatsu, Hamamatsu City, Japan), blood vessels, highlighted by anti-CD34 immunostaining, were manually marked on the digital vascular endothelial growth factor receptor (VEGFR) slides by a pathologist using the NDP.view software. The tissue core diameter is 600 µm. ( B ) The delineated regions were imported into the Visiopharm software to quantify the VEGF, VEGFR-1 or VEGFR-2 expression levels using the labelling index (LI), i.e., the percentage of the immunoreactive area within the vessel area.

    Techniques Used: Microarray, Immunostaining, Software, Expressing

    Prognostic value of VEGFR-1 and VEGFR-2 expression in EC of CRC: ( A ) Metastasis-free survival curves of patients dichotomized based on VEGFR-1 LI ≤ (solid line) or > (dotted line) 5% ( p = 0.063). ( B ) Overall survival curves of patients dichotomized based on VEGFR-1 LI ≤ (solid line) or > (dotted line) 5% ( p = 0.013). ( C ) Metastasis-free survival curves of patients dichotomized based on VEGFR-2 LI ≤ (solid line) or > (dotted line) 33% ( p = 0.002). Complete and censured data are shown as dots and crosses, respectively.
    Figure Legend Snippet: Prognostic value of VEGFR-1 and VEGFR-2 expression in EC of CRC: ( A ) Metastasis-free survival curves of patients dichotomized based on VEGFR-1 LI ≤ (solid line) or > (dotted line) 5% ( p = 0.063). ( B ) Overall survival curves of patients dichotomized based on VEGFR-1 LI ≤ (solid line) or > (dotted line) 5% ( p = 0.013). ( C ) Metastasis-free survival curves of patients dichotomized based on VEGFR-2 LI ≤ (solid line) or > (dotted line) 33% ( p = 0.002). Complete and censured data are shown as dots and crosses, respectively.

    Techniques Used: Expressing

    22) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    23) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    24) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    25) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    26) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    27) Product Images from "Stimulation of lymphangiogenesis via VEGFR-3 inhibits chronic skin inflammation"

    Article Title: Stimulation of lymphangiogenesis via VEGFR-3 inhibits chronic skin inflammation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20100559

    Systemic inhibition of VEGFR-1 does not reduce chronic skin inflammation. (A) Hemizygous K14-VEGF-A Tg mice ( n = 20) were painted and challenged as described in Fig. 1 . Starting at study day 7, mice received five i.p. injections of mAb MF1 (anti–VEGFR-1, ■), MF1 + DC101 (anti-VEGFR-1+2, ▲), DC101 + mF4-31C1 (anti–VEGFR-2+3, ○), or control rat IgG (⋄, n = 5 per group) every 3 d. Treatment with MF1 did not affect the chronic inflammatory response, whereas combined treatment with DC101 + MF1 and with DC101 + mF4-31C1 potently reduced skin inflammatory ear swelling. Data represent mean ± SEM. Two independent experiments were performed. (B) H E stains of mouse ears at study day 21 after oxazolone challenge revealed edema formation, epidermal thickening, and inflammatory cell infiltration in the IgG control group and after anti–VEGFR-1 mAb treatment. Anti–VEGFR-1+2 and anti–VEGFR-2+3 treatment largely normalized skin architecture. One ear half is shown. (C and D) Immunofluorescence stainings and quantification of BrdU + cells show similar numbers of proliferating cells in the ear skin after anti–VEGFR-1 (R1, anti-R1) mAb treatment compared with control IgG-injected mice. Systemic inhibition of VEGFR-1+2 (R1+2, anti-R1+2) or VEGFR-2+3 (R2+3, anti-R2+3) reduced the number of BrdU + cells. Data represent mean ± SD. *, P
    Figure Legend Snippet: Systemic inhibition of VEGFR-1 does not reduce chronic skin inflammation. (A) Hemizygous K14-VEGF-A Tg mice ( n = 20) were painted and challenged as described in Fig. 1 . Starting at study day 7, mice received five i.p. injections of mAb MF1 (anti–VEGFR-1, ■), MF1 + DC101 (anti-VEGFR-1+2, ▲), DC101 + mF4-31C1 (anti–VEGFR-2+3, ○), or control rat IgG (⋄, n = 5 per group) every 3 d. Treatment with MF1 did not affect the chronic inflammatory response, whereas combined treatment with DC101 + MF1 and with DC101 + mF4-31C1 potently reduced skin inflammatory ear swelling. Data represent mean ± SEM. Two independent experiments were performed. (B) H E stains of mouse ears at study day 21 after oxazolone challenge revealed edema formation, epidermal thickening, and inflammatory cell infiltration in the IgG control group and after anti–VEGFR-1 mAb treatment. Anti–VEGFR-1+2 and anti–VEGFR-2+3 treatment largely normalized skin architecture. One ear half is shown. (C and D) Immunofluorescence stainings and quantification of BrdU + cells show similar numbers of proliferating cells in the ear skin after anti–VEGFR-1 (R1, anti-R1) mAb treatment compared with control IgG-injected mice. Systemic inhibition of VEGFR-1+2 (R1+2, anti-R1+2) or VEGFR-2+3 (R2+3, anti-R2+3) reduced the number of BrdU + cells. Data represent mean ± SD. *, P

    Techniques Used: Inhibition, Mouse Assay, Immunofluorescence, Injection

    Systemic blockade of VEGFR-2 results in normalized epidermal differentiation and reduced inflammatory skin infiltration. (A and B) Immunofluorescence analyses of ear skin sections from normal wild-type mice show that the hyperproliferation-associated keratin 6 is only faintly expressed in the normal interfollicular epidermis and is largely restricted to the hair follicle (A). Loricrin, a marker of terminal epidermal differentiation, is restricted to the upper granular layer in normal mouse skin (B). At day 21 of chronic skin inflammation in control IgG-treated mice, keratin 6 and loricrin show a much broader expression pattern. Inhibition of VEGFR-2 (anti-R2), alone or in combination with inhibition of VEGFR-1 or VEGFR-3, largely normalized keratin 6 and loricrin expression patterns. Treatment with anti–VEGFR-1 or anti–VEGR-3 showed no major effect. (C and E) CD11b + cells are rarely found in normal mouse skin (wt). The number of CD11b + cells was increased in the inflamed skin of control IgG-treated mice and was decreased after the inhibition of VEGFR-2, VEGFR-1+2, or VEGFR-2+3 but not after inhibition of VEGFR-1. Inhibition of VEGFR-3 resulted in enhanced numbers of CD11b + cells. (D and E) Inhibition of VEGFR-2, VEGFR-1+2, and VEGFR-2+3 strongly decreased the number of intraepidermal CD8 + T-lymphocytes, whereas the inhibition of VEGFR-1 or VEGFR-3 had no effect, as compared with IgG-treated mice. Bars, 100 µm. (E) Computer-assisted quantification of the numbers of CD11b + and CD8 + cells per millimeter of epidermal basement membrane. n = 5 mice per group. Two independent experiments were performed. Data represent mean ± SD. *, P
    Figure Legend Snippet: Systemic blockade of VEGFR-2 results in normalized epidermal differentiation and reduced inflammatory skin infiltration. (A and B) Immunofluorescence analyses of ear skin sections from normal wild-type mice show that the hyperproliferation-associated keratin 6 is only faintly expressed in the normal interfollicular epidermis and is largely restricted to the hair follicle (A). Loricrin, a marker of terminal epidermal differentiation, is restricted to the upper granular layer in normal mouse skin (B). At day 21 of chronic skin inflammation in control IgG-treated mice, keratin 6 and loricrin show a much broader expression pattern. Inhibition of VEGFR-2 (anti-R2), alone or in combination with inhibition of VEGFR-1 or VEGFR-3, largely normalized keratin 6 and loricrin expression patterns. Treatment with anti–VEGFR-1 or anti–VEGR-3 showed no major effect. (C and E) CD11b + cells are rarely found in normal mouse skin (wt). The number of CD11b + cells was increased in the inflamed skin of control IgG-treated mice and was decreased after the inhibition of VEGFR-2, VEGFR-1+2, or VEGFR-2+3 but not after inhibition of VEGFR-1. Inhibition of VEGFR-3 resulted in enhanced numbers of CD11b + cells. (D and E) Inhibition of VEGFR-2, VEGFR-1+2, and VEGFR-2+3 strongly decreased the number of intraepidermal CD8 + T-lymphocytes, whereas the inhibition of VEGFR-1 or VEGFR-3 had no effect, as compared with IgG-treated mice. Bars, 100 µm. (E) Computer-assisted quantification of the numbers of CD11b + and CD8 + cells per millimeter of epidermal basement membrane. n = 5 mice per group. Two independent experiments were performed. Data represent mean ± SD. *, P

    Techniques Used: Immunofluorescence, Mouse Assay, Marker, Expressing, Inhibition

    Inflammatory lymphangiogenesis is inhibited by blockade of VEGFR-2 or of VEGFR-3. (A) Quantitative image analyses of lymphatic vessels (LV; LYVE-1 + , green) revealed a significantly increased number and size of lymphatic vessels in control IgG injected mice at day 21 after induction of inflammation, as compared with normal wild-type (wt) mice. The mean number of lymphatic vessels was reduced after anti–VEGFR-2 (R2) and after anti–VEGFR-3 (R3) treatment but not after systemic inhibition of VEGFR-1 (R1). Combined inhibition of VEGFR-1+2 (1+2) or VEGFR-2+3 (2+3) also reduced lymphatic vessel numbers. Systemic inhibition of VEGFR-2, VEGFR-1+2, and VEGFR-2+3 resulted in a reduced mean size of lymphatic vessels. n = 5 mice per group. Two independent experiments were performed. Data represent mean ± SD. ‡, P
    Figure Legend Snippet: Inflammatory lymphangiogenesis is inhibited by blockade of VEGFR-2 or of VEGFR-3. (A) Quantitative image analyses of lymphatic vessels (LV; LYVE-1 + , green) revealed a significantly increased number and size of lymphatic vessels in control IgG injected mice at day 21 after induction of inflammation, as compared with normal wild-type (wt) mice. The mean number of lymphatic vessels was reduced after anti–VEGFR-2 (R2) and after anti–VEGFR-3 (R3) treatment but not after systemic inhibition of VEGFR-1 (R1). Combined inhibition of VEGFR-1+2 (1+2) or VEGFR-2+3 (2+3) also reduced lymphatic vessel numbers. Systemic inhibition of VEGFR-2, VEGFR-1+2, and VEGFR-2+3 resulted in a reduced mean size of lymphatic vessels. n = 5 mice per group. Two independent experiments were performed. Data represent mean ± SD. ‡, P

    Techniques Used: Injection, Mouse Assay, Inhibition

    Expression of VEGFR and IL-23a mRNA after antibody injections. TaqMan-based real-time RT-PCR analyses were performed on whole ear skin extracts after 14 d of antibody treatment (study day 21) in K14-VEGF-A Tg mice. VEGFR-1, VEGFR-2, VEGFR-3, and the IL-23–specific subunit p19 (IL-23a) were significantly up-regulated in inflamed IgG-injected mouse skin compared with untreated wild-type mice. The modulation of mRNA transcript levels after the corresponding antibody injections is shown. n = 5 mice per group. Two independent experiments were performed. Data represent mean ± SD. ‡, P
    Figure Legend Snippet: Expression of VEGFR and IL-23a mRNA after antibody injections. TaqMan-based real-time RT-PCR analyses were performed on whole ear skin extracts after 14 d of antibody treatment (study day 21) in K14-VEGF-A Tg mice. VEGFR-1, VEGFR-2, VEGFR-3, and the IL-23–specific subunit p19 (IL-23a) were significantly up-regulated in inflamed IgG-injected mouse skin compared with untreated wild-type mice. The modulation of mRNA transcript levels after the corresponding antibody injections is shown. n = 5 mice per group. Two independent experiments were performed. Data represent mean ± SD. ‡, P

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

    28) Product Images from "Paracrine Modulation of CXCR4 by IGF-1 and VEGF: Implications for Choroidal Neovascularization"

    Article Title: Paracrine Modulation of CXCR4 by IGF-1 and VEGF: Implications for Choroidal Neovascularization

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.09-4137

    Analysis of mRNA levels in the posterior cups. Neural retinas were removed from posterior cups; the retinal pigment epithelium/choroid complex was pooled to analyze the levels of VEGF-A, VEGFR-1, VEGFR-2, SDF-1, CXCR4, CXCR7, IGF-1, and IGF-1R in response
    Figure Legend Snippet: Analysis of mRNA levels in the posterior cups. Neural retinas were removed from posterior cups; the retinal pigment epithelium/choroid complex was pooled to analyze the levels of VEGF-A, VEGFR-1, VEGFR-2, SDF-1, CXCR4, CXCR7, IGF-1, and IGF-1R in response

    Techniques Used:

    Percentage of cells expressing VEGFR-1 or VEGFR-2 in CD34 + EPCs after SDF-1 exposure. CD34 + EPCs were exposed to 0.1 nM SDF-1, 100 nM SDF-1 or medium alone (control) for 15 minutes, 1 hour, 4 hours, and 6 hours. Cells were stained with anti-VEGFR-1 or
    Figure Legend Snippet: Percentage of cells expressing VEGFR-1 or VEGFR-2 in CD34 + EPCs after SDF-1 exposure. CD34 + EPCs were exposed to 0.1 nM SDF-1, 100 nM SDF-1 or medium alone (control) for 15 minutes, 1 hour, 4 hours, and 6 hours. Cells were stained with anti-VEGFR-1 or

    Techniques Used: Expressing, Staining

    29) Product Images from "Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib"

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14755

    Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the susceptibility to vemurafenib of M14‐VR and M14 cells. A and B, M14‐VR (A) or M14 (B) cells (1000/well) were seeded into 96‐well plates and the day after transfected with 10 nmol/L siVEGFR‐1 or siCTR and treated with graded concentrations of vemurafenib. After 5 d of culture, cell growth was analysed by MTS assay. Data are the mean of three independent experiments. Statistical analysis by two‐tailed Student's t test: * P

    Techniques Used: Transfection, MTS Assay, Two Tailed Test

    Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Expression of VEGFR‐1 in melanoma cells with proliferative or invasive phenotypes and inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by M14‐VR melanoma cells in response to PlGF or VEGF‐A. A, HOPP analysis based on VEGFR‐1 expression levels was carried out using gene expression data sets including 189 melanoma cell lines and short‐term cultures, of which 100 are characterized by a proliferative phenotype and 89 by an invasive phenotype. 33 Mean VEGFR‐1 transcript levels for proliferative (PRO) melanomas were compared with those of invasive melanomas (INV) and expressed as normalized signal intensity. Analysis of the 222033_s_at probeset for VEGFR‐1:3.9‐fold significant difference; statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Two Tailed Test

    Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Enforced VEGFR‐1 expression in M14 melanoma cells increases invasiveness and reduces sensitivity to vemurafenib. A, VEGFR‐1 protein levels in M14 cells transfected with control (M14‐C) or VEGFR‐1 expressing (M14‐MF5) vectors were analysed by immunoblotting using antibodies against human VEGFR‐1 or β‐tubulin as loading control. The VEGFR‐1 protein has an expected molecular weight of 150 kD for the unmodified polypeptide and of 180‐185 kD for the glycosylated mature form. Positive control (Pos. CTR): glioblastoma cells transfected with the pBLAS49.2/VEGFR‐1 plasmid overexpressing the receptor. 42 B, Western blot of phosphorylated Erk (pErk) and total Erk in M14, M14‐C and M14‐MF5 cells; β‐tubulin (β‐Tub) was used as loading control. Histogram represents the densitometric quantification of band intensities, expressed as pErk/Erk ratio relative to M14 non transfected cells, after normalization for β‐tubulin expression. Normalized pErk1/Erk protein ratio in M14 cells was considered equal to 1.Data are the mean ± SD of three independent experiments. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Expressing, Transfection, Molecular Weight, Positive Control, Plasmid Preparation, Western Blot, Two Tailed Test

    Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P
    Figure Legend Snippet: Influence of VEGFR‐1 silencing on the acquisition of resistance to vemurafenib by A375 cells. A, A375 cells were transfected with 10 nmol/L siVEGFR‐1 or siCTR and after three days total RNA was extracted and membrane VEGFR‐1 (mVEGFR‐1) transcript levels were assessed by qRT‐PCR analysis. Data are the mean of three independent determinations. Statistical analysis by two‐tailed Student's t test: *** P

    Techniques Used: Transfection, Quantitative RT-PCR, Two Tailed Test

    Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P
    Figure Legend Snippet: Characterization of human melanoma cell lines sensitive or resistant to vemurafenib for the production of VEGF‐A and PlGF and expression of VEGFR‐1. VEGF‐A (A) and PlGF (B) secretion was quantified by ELISA (mean ± SD; n = 3). Each value represents the arithmetic mean of three independent experiments performed with triplicate samples. C, The expression of VEGFR‐1 transcript was assessed by qRT‐PCR analysis utilizing the human melanoma GR‐Mel cell line as positive control. The results are expressed as relative mRNA and are the mean ± SD of three (A375 lines) or two (M14 lines) independent determinations with duplicate samples. Data were referred to the VEGFR‐1 negative M14 bulk cell line, to which the arbitrary value of 1 was assigned. Statistical analysis by two‐tailed Student's t test: resistant vs sensitive cells: *** P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Positive Control, Two Tailed Test

    Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P
    Figure Legend Snippet: Inhibitory effect of the anti‐VEGFR‐1 mAb D16F7 on ECM invasion by A375‐VR melanoma cells in response to PlGF. A, ECM invasion of A375‐VR cells (2x10 5 cells/chamber, 4 h incubation) induced by PlGF (50 ng/mL) in the absence or presence of 5 µg/mL D16F7 mAb. BSA, non‐stimulated cells. Histograms represent the arithmetic mean ± SD of invading cells/microscopic field from three independent experiments. Statistical analysis was performed by Kruskal‐Wallis followed by Dunn's test for multiple comparison: P

    Techniques Used: Incubation

    Related Articles

    Transfection:

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib
    Article Snippet: .. M14‐VR cells silenced for VEGFR‐1 showed a significant increase of susceptibility to vemurafenib compared with siCTR transfected cells (Figure A). .. In these experimental conditions, the IC50 value of M14‐VR cells transfected with siCTR resulted 31 ± 3.5 µmol/L, while that of M14‐VR cells silenced for VEGFR‐1 was 15.7 ± 1.0 µmol/L.

    Amplification:

    Article Title: Downregulation of flt-1 and HIF-1α Gene Expression by Some Antioxidants in Rats Under Sodium Nitrite–Induced Hypoxic Stress
    Article Snippet: .. Real-time PCR for flt-1 and hypoxia inducible factor 1α determinations Real-time PCR amplification and analysis were performed using an Applied Biosystems thermocycler with software version 3.1 (StepOne). .. The reaction contained SYBR Green Master Mix (Applied Biosystems).

    Synthesized:

    Article Title: Toxicological Evaluation of SiO2 Nanoparticles by Zebrafish Embryo Toxicity Test
    Article Snippet: .. The first-strand cDNA was synthesized from 200 ng of total RNA using the SuperScript III cDNA Synthesis Kit (Thermo Fisher Scientific) with oligo dT primers (Thermo Fisher Scientific). cDNA (n = 5 in each group) was subjected to quantitative polymerase chain reaction (PCR) analysis with FastStart Universal Probe Master Mix (Roche, Basel, Switzerland) and primers for VEGFa and VEGF receptors (VEGFR1; Flt-1: Fms-like tyrosine kinase and VEGFR2; KDR: kinase insert domain receptor) using an ABI 7300 Real-Time PCR system (Thermo Fisher Scientific), as described previously [ ].The gene expression level was normalized to that of β-actin in the same cDNA. ..

    Real-time Polymerase Chain Reaction:

    Article Title: Toxicological Evaluation of SiO2 Nanoparticles by Zebrafish Embryo Toxicity Test
    Article Snippet: .. The first-strand cDNA was synthesized from 200 ng of total RNA using the SuperScript III cDNA Synthesis Kit (Thermo Fisher Scientific) with oligo dT primers (Thermo Fisher Scientific). cDNA (n = 5 in each group) was subjected to quantitative polymerase chain reaction (PCR) analysis with FastStart Universal Probe Master Mix (Roche, Basel, Switzerland) and primers for VEGFa and VEGF receptors (VEGFR1; Flt-1: Fms-like tyrosine kinase and VEGFR2; KDR: kinase insert domain receptor) using an ABI 7300 Real-Time PCR system (Thermo Fisher Scientific), as described previously [ ].The gene expression level was normalized to that of β-actin in the same cDNA. ..

    Article Title: Downregulation of flt-1 and HIF-1α Gene Expression by Some Antioxidants in Rats Under Sodium Nitrite–Induced Hypoxic Stress
    Article Snippet: .. Real-time PCR for flt-1 and hypoxia inducible factor 1α determinations Real-time PCR amplification and analysis were performed using an Applied Biosystems thermocycler with software version 3.1 (StepOne). .. The reaction contained SYBR Green Master Mix (Applied Biosystems).

    Concentration Assay:

    Article Title: A General Approach for Receptor and Antibody-Targeted Detection of Native Proteins utilizing Split-Luciferase Reassembly
    Article Snippet: .. Flt-1 was refolded overnight at 4 °C in 2 L of refolding buffer (0.3 M urea, 10 mM Tris-HCl, 100 mM Na2 HPO4 , 5 mM cysteine and 1 mM cystine) system in a 3 kDa MWCO dialysis snake-skin tubing (Pierce) at a final concentration of 50 – 100 µg mL−1 . .. After refolding, Flt-1 was subsequently purified in phosphate buffered saline (pH 7.4) using an analytical Superdex™ 75 column (Pharmacia Biotech).

    other:

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib
    Article Snippet: 3.3 VEGFR‐1 silencing counteracts the emergence of resistance in sensitive cells and increases sensitivity to vemurafenib in resistant melanoma cells To investigate the role of VEGFR‐1 in the acquisition of a vemurafenib‐resistant phenotype, A375 cells, which express basal levels of VEGFR‐1, were transiently silenced for the receptor and exposed to vemurafenib.

    Article Title: Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib, et al. Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib
    Article Snippet: Interestingly, VEGFR‐1 blockade by D16F7 mAb reduces ECM invasion triggered by VEGF‐A and PlGF, supporting the hypothesis that up‐regulation of VEGFR‐1 might contribute to tumour progression and spreading of melanoma after acquisition of a drug‐resistant phenotype.

    Expressing:

    Article Title: Toxicological Evaluation of SiO2 Nanoparticles by Zebrafish Embryo Toxicity Test
    Article Snippet: .. The first-strand cDNA was synthesized from 200 ng of total RNA using the SuperScript III cDNA Synthesis Kit (Thermo Fisher Scientific) with oligo dT primers (Thermo Fisher Scientific). cDNA (n = 5 in each group) was subjected to quantitative polymerase chain reaction (PCR) analysis with FastStart Universal Probe Master Mix (Roche, Basel, Switzerland) and primers for VEGFa and VEGF receptors (VEGFR1; Flt-1: Fms-like tyrosine kinase and VEGFR2; KDR: kinase insert domain receptor) using an ABI 7300 Real-Time PCR system (Thermo Fisher Scientific), as described previously [ ].The gene expression level was normalized to that of β-actin in the same cDNA. ..

    Polymerase Chain Reaction:

    Article Title: Toxicological Evaluation of SiO2 Nanoparticles by Zebrafish Embryo Toxicity Test
    Article Snippet: .. The first-strand cDNA was synthesized from 200 ng of total RNA using the SuperScript III cDNA Synthesis Kit (Thermo Fisher Scientific) with oligo dT primers (Thermo Fisher Scientific). cDNA (n = 5 in each group) was subjected to quantitative polymerase chain reaction (PCR) analysis with FastStart Universal Probe Master Mix (Roche, Basel, Switzerland) and primers for VEGFa and VEGF receptors (VEGFR1; Flt-1: Fms-like tyrosine kinase and VEGFR2; KDR: kinase insert domain receptor) using an ABI 7300 Real-Time PCR system (Thermo Fisher Scientific), as described previously [ ].The gene expression level was normalized to that of β-actin in the same cDNA. ..

    Software:

    Article Title: Downregulation of flt-1 and HIF-1α Gene Expression by Some Antioxidants in Rats Under Sodium Nitrite–Induced Hypoxic Stress
    Article Snippet: .. Real-time PCR for flt-1 and hypoxia inducible factor 1α determinations Real-time PCR amplification and analysis were performed using an Applied Biosystems thermocycler with software version 3.1 (StepOne). .. The reaction contained SYBR Green Master Mix (Applied Biosystems).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher vegfa
    Western blot data of E 19 hypoxia signaling proteins and matrix metalloproteinases of sham and TXA 2 analog exposed (FGR) placentae separated out by sex. (A) Quantification of HIF-1α, HIF-2α, HIF-1β, <t>PHD2,</t> VHL, and <t>VEGFA</t> protein abundance is depicted. (B) Quantification of gelatinases (MMP2, MMP9), membrane-type MMPs (MMP14, MMP15, MMP16, MMP17, MMP24, MMP25), and EMMPRIN (CD147) is depicted. a denotes statistical significance between sham females (SF) and sham males (SM) at P
    Vegfa, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vegfa/product/Thermo Fisher
    Average 99 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    vegfa - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    96
    Thermo Fisher vegfr 2
    VEGF-induced activation of c-Src, Akt, but not <t>VEGFR-2,</t> PLCγ-1 and ERK1/2 requires NADPH oxidase-derived ROS. (A) Protein extracts from HCAEC transfected with control siRNA (Scram-si) or si-p47 phox were subject to Western blots as described in Materials and methods . Serum-starved HCAEC were treated with VEGF (50 ng/ml) for the times indicated. Membranes were sequentially blotted, stripped and re-probed with the phospho-specific antibodies as shown. Blots shown are representative of three independent experiments. (B) Same as in (A) except, the membranes were probed for phosphorylation of Y1175 VEGFR-2, Y783 PLCγ-1 and p42/44 ERK1/2. Anti-β-actin antibody was used as loading control.
    Vegfr 2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vegfr 2/product/Thermo Fisher
    Average 96 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    vegfr 2 - by Bioz Stars, 2020-09
    96/100 stars
      Buy from Supplier

    96
    Thermo Fisher antibodies against vegf
    Heterogeneous <t>VEGF,</t> <t>VEGFR-1</t> and VEGFR-2 expression in colorectal cancer (CRC) endothelial cells (ECs): VEGF expression in ECs was heterogeneous with an labelling index (LI) range from 10.9 to 90% ( A , D ); Anti-VEGFR-1 immunostaining shows rare positivity with an LI range from nearly 0 ( B ) to 20% ( E ) in endothelial cells. VEGFR-2 shows a much wider immunostaining, with an LI range from 10 ( C ) to 72% ( F ). (Magnification: 400×). The arrows are pointed at vessels.
    Antibodies Against Vegf, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against vegf/product/Thermo Fisher
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antibodies against vegf - by Bioz Stars, 2020-09
    96/100 stars
      Buy from Supplier

    Image Search Results


    Western blot data of E 19 hypoxia signaling proteins and matrix metalloproteinases of sham and TXA 2 analog exposed (FGR) placentae separated out by sex. (A) Quantification of HIF-1α, HIF-2α, HIF-1β, PHD2, VHL, and VEGFA protein abundance is depicted. (B) Quantification of gelatinases (MMP2, MMP9), membrane-type MMPs (MMP14, MMP15, MMP16, MMP17, MMP24, MMP25), and EMMPRIN (CD147) is depicted. a denotes statistical significance between sham females (SF) and sham males (SM) at P

    Journal: Biology of Reproduction

    Article Title: Effects of excess thromboxane A2 on placental development and nutrient transporters in a Mus musculus model of fetal growth restriction

    doi: 10.1093/biolre/ioy006

    Figure Lengend Snippet: Western blot data of E 19 hypoxia signaling proteins and matrix metalloproteinases of sham and TXA 2 analog exposed (FGR) placentae separated out by sex. (A) Quantification of HIF-1α, HIF-2α, HIF-1β, PHD2, VHL, and VEGFA protein abundance is depicted. (B) Quantification of gelatinases (MMP2, MMP9), membrane-type MMPs (MMP14, MMP15, MMP16, MMP17, MMP24, MMP25), and EMMPRIN (CD147) is depicted. a denotes statistical significance between sham females (SF) and sham males (SM) at P

    Article Snippet: We incubated membranes with the following antibodies: HIF-1α (prod#PA116601, ThermoFisher Scientific, Waltham, MA) at 1:200, HIF1β (prod#PA316508, ThermoFisher Scientific) at 1:200, HIF2α (prod#PA116510, ThermoFisher Scientific) at 1:300, PHD2 (prod#PA116524, ThermoFisher Scientific) at 1:300, VHL (prod#PA527322, ThermoFisher Scientific) at 1:200, VEGFA (prod#PA516754, ThermoFisher Scientific) at 1:100, MMP2 (prod#PA516504, ThermoFisher Scientific) at 1:200, MMP9 (prod#PA513199, ThermoFisher Scientific) at 1:200, MMP14 (prod#PA516514, ThermoFisher Scientific) at 1:100, MMP15 (prod#PA513184, ThermoFisher Scientific) at 1:400, MMP16 (prod#PA528966, ThermoFisher Scientific) at 1:400, MMP17 (prod#PA528219, ThermoFisher Scientific) at 1:200, MMP24 (prod#PA551333, ThermoFisher Scientific) at 1:100, MMP25 (prod#PA513191, ThermoFisher Scientific) at 1:100, CD147 (prod#11989–1-AP, Proteintech) at 1:100; GLUT1 (prod#07–1401, Millipore) at 1:500, GLUT3 (prod#ab41525, Abcam, Cambridge, MA) at 1:500, SNAT1 (prod#PA542420, ThermoFisher Scientific) at 1:500, SNAT2 (prod#SC-67081, Santa Cruz Biotech, Santa Cruz, CA) at 1:400, SNAT4 (prod#SC-67085, Santa Cruz Biotech) at 1:200, FATP2 (prod#ab175373, Abcam) at 1:200, FATP4 (prod#ab199719, Abcam) at 1:500, and CD36 (prod#ab133625, Abcam) at 1:500 for 1 h at RT (see full list of antibodies under ).

    Techniques: Western Blot

    VEGF-induced activation of c-Src, Akt, but not VEGFR-2, PLCγ-1 and ERK1/2 requires NADPH oxidase-derived ROS. (A) Protein extracts from HCAEC transfected with control siRNA (Scram-si) or si-p47 phox were subject to Western blots as described in Materials and methods . Serum-starved HCAEC were treated with VEGF (50 ng/ml) for the times indicated. Membranes were sequentially blotted, stripped and re-probed with the phospho-specific antibodies as shown. Blots shown are representative of three independent experiments. (B) Same as in (A) except, the membranes were probed for phosphorylation of Y1175 VEGFR-2, Y783 PLCγ-1 and p42/44 ERK1/2. Anti-β-actin antibody was used as loading control.

    Journal: PLoS ONE

    Article Title: Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

    doi: 10.1371/journal.pone.0028454

    Figure Lengend Snippet: VEGF-induced activation of c-Src, Akt, but not VEGFR-2, PLCγ-1 and ERK1/2 requires NADPH oxidase-derived ROS. (A) Protein extracts from HCAEC transfected with control siRNA (Scram-si) or si-p47 phox were subject to Western blots as described in Materials and methods . Serum-starved HCAEC were treated with VEGF (50 ng/ml) for the times indicated. Membranes were sequentially blotted, stripped and re-probed with the phospho-specific antibodies as shown. Blots shown are representative of three independent experiments. (B) Same as in (A) except, the membranes were probed for phosphorylation of Y1175 VEGFR-2, Y783 PLCγ-1 and p42/44 ERK1/2. Anti-β-actin antibody was used as loading control.

    Article Snippet: Colocalization of VEGFR-2 with c-Src or EEA1-positive endosome by immunofluorescence HCAEC were plated on fibronectin coated glass-bottom chamber slides (Lab-Tek II, Thermo Scientific) and starved overnight in 0.2% FBS containing EBM-2.

    Techniques: Activation Assay, Derivative Assay, Transfection, Western Blot

    VEGF-induced interaction between VEGFR-2 and c-Src requires NADPH oxidase-derived ROS. (A) Co-immunoprecipitation (co-IP) assay using 1.2 mg protein lysates of HCAEC that were transfected with Scram-si or si-p47 phox and treated without or with VEGF (50 ng/ml for 5 min). IP was carried out using anti-VEGFR-2 antibody followed by immunoblotting using anti-c-Src (upper panel) and anti-VEGFR-2 (lower panel) antibodies. (B) Quantitative analyses of VEGFR-2-bound c-Src. Bar graphs show quantitative densitometric analysis of three independent experiments using NIH image J (-fold change expressed in mean ± S.E.M.). * p

    Journal: PLoS ONE

    Article Title: Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

    doi: 10.1371/journal.pone.0028454

    Figure Lengend Snippet: VEGF-induced interaction between VEGFR-2 and c-Src requires NADPH oxidase-derived ROS. (A) Co-immunoprecipitation (co-IP) assay using 1.2 mg protein lysates of HCAEC that were transfected with Scram-si or si-p47 phox and treated without or with VEGF (50 ng/ml for 5 min). IP was carried out using anti-VEGFR-2 antibody followed by immunoblotting using anti-c-Src (upper panel) and anti-VEGFR-2 (lower panel) antibodies. (B) Quantitative analyses of VEGFR-2-bound c-Src. Bar graphs show quantitative densitometric analysis of three independent experiments using NIH image J (-fold change expressed in mean ± S.E.M.). * p

    Article Snippet: Colocalization of VEGFR-2 with c-Src or EEA1-positive endosome by immunofluorescence HCAEC were plated on fibronectin coated glass-bottom chamber slides (Lab-Tek II, Thermo Scientific) and starved overnight in 0.2% FBS containing EBM-2.

    Techniques: Derivative Assay, Co-Immunoprecipitation Assay, Transfection

    c-Src and VEGFR-2 are oxidized in VEGF-treated HCAEC in the presence of NADPH oxidase-derived ROS. (A) Schematic presentation depicting cysteinyl-labeling assay to determine oxidative modification in intracellular proteins. Non-oxidized protein thiols are alkyalated by IAA, oxidized thiols are reduced back to SH-moiety by DTT and subsequently biotinylated by IAP. Biotinylated proteins are then pulled-down by streptavidin-agarose followed by Western blots. (B) Upper panel: cysteinyl labeling assay to Identify thiol oxidation of proteins in VEGF-treated (50 ng/ml for 2 mins) HCAEC lysates using biotinylated IAP probe. HCAEC were transfected with Scram-si or si-p47 phox as indicated. After cell lysis in the presence of IAA followed by DTT treatment and IAP labeling, 1.5 mg biotinylated protein lysates were subject to immunoprecipitation using Streptavidin-agarose beads and immunoblotted using anti-VEGFR-2 and anti-c-Src antibodies. Lower panel: Western blot for VEGFR-2 using 50 µg of parallel HCAEC lysates as loading control. (B) Quantitative analyses of oxidized VEGFR-2 (upper panel) and c-Src (lower panel). Bar graph shows quantitative densitometric analysis of three independent cysteinyl labeling assays (as in A) using NIH J image (-fold change expressed in mean ± S.E.M.). * p

    Journal: PLoS ONE

    Article Title: Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

    doi: 10.1371/journal.pone.0028454

    Figure Lengend Snippet: c-Src and VEGFR-2 are oxidized in VEGF-treated HCAEC in the presence of NADPH oxidase-derived ROS. (A) Schematic presentation depicting cysteinyl-labeling assay to determine oxidative modification in intracellular proteins. Non-oxidized protein thiols are alkyalated by IAA, oxidized thiols are reduced back to SH-moiety by DTT and subsequently biotinylated by IAP. Biotinylated proteins are then pulled-down by streptavidin-agarose followed by Western blots. (B) Upper panel: cysteinyl labeling assay to Identify thiol oxidation of proteins in VEGF-treated (50 ng/ml for 2 mins) HCAEC lysates using biotinylated IAP probe. HCAEC were transfected with Scram-si or si-p47 phox as indicated. After cell lysis in the presence of IAA followed by DTT treatment and IAP labeling, 1.5 mg biotinylated protein lysates were subject to immunoprecipitation using Streptavidin-agarose beads and immunoblotted using anti-VEGFR-2 and anti-c-Src antibodies. Lower panel: Western blot for VEGFR-2 using 50 µg of parallel HCAEC lysates as loading control. (B) Quantitative analyses of oxidized VEGFR-2 (upper panel) and c-Src (lower panel). Bar graph shows quantitative densitometric analysis of three independent cysteinyl labeling assays (as in A) using NIH J image (-fold change expressed in mean ± S.E.M.). * p

    Article Snippet: Colocalization of VEGFR-2 with c-Src or EEA1-positive endosome by immunofluorescence HCAEC were plated on fibronectin coated glass-bottom chamber slides (Lab-Tek II, Thermo Scientific) and starved overnight in 0.2% FBS containing EBM-2.

    Techniques: Derivative Assay, Labeling, Modification, Western Blot, Transfection, Lysis, Immunoprecipitation

    Proposed model: thiol oxidation may help propagate signal transduction from VEGFR-2 to downstream c-Src. (A) VEGF activation of VEGFR-2 and downstream PLCγ-1-ERK1/2 signaling pathway appears to be independent of ROS levels in ECs. (B) VEGF induces NADPH oxidase-derived ROS, which in turn oxidizes VEGFR-2 and c-Src. Thiol oxidation of these two tyrosine kinases appears to correlate with VEGF-induced activation of c-Src, and also with the sub-cellular colocalization and interaction between VEGFR-2 and c-Src. Dependence of VEGF-induced thiol oxidation and activation of c-Src on NADPH oxidase-derived ROS render downstream activation of PI3K-Akt redox-sensitive in HCAEC. In this model, VEGFR-2 and/or c-Src act as endothelial redox-sensors that determine whether downstream PI3K-Akt signaling pathway should be activated or not.

    Journal: PLoS ONE

    Article Title: Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

    doi: 10.1371/journal.pone.0028454

    Figure Lengend Snippet: Proposed model: thiol oxidation may help propagate signal transduction from VEGFR-2 to downstream c-Src. (A) VEGF activation of VEGFR-2 and downstream PLCγ-1-ERK1/2 signaling pathway appears to be independent of ROS levels in ECs. (B) VEGF induces NADPH oxidase-derived ROS, which in turn oxidizes VEGFR-2 and c-Src. Thiol oxidation of these two tyrosine kinases appears to correlate with VEGF-induced activation of c-Src, and also with the sub-cellular colocalization and interaction between VEGFR-2 and c-Src. Dependence of VEGF-induced thiol oxidation and activation of c-Src on NADPH oxidase-derived ROS render downstream activation of PI3K-Akt redox-sensitive in HCAEC. In this model, VEGFR-2 and/or c-Src act as endothelial redox-sensors that determine whether downstream PI3K-Akt signaling pathway should be activated or not.

    Article Snippet: Colocalization of VEGFR-2 with c-Src or EEA1-positive endosome by immunofluorescence HCAEC were plated on fibronectin coated glass-bottom chamber slides (Lab-Tek II, Thermo Scientific) and starved overnight in 0.2% FBS containing EBM-2.

    Techniques: Transduction, Activation Assay, Derivative Assay, Activated Clotting Time Assay

    VEGF induces subcellular co-localization of c-Src and internalized VEGFR-2 in an ROS-dependent manner. HCAEC transfected with control (Scram-si) (A) or si-p47 phox (B) were immunofluorescently double labeled for internalized VEGFR-2 (green) and c-Src (red). VEGFR-2 on HCAEC was labeled with single chain E-tagged antibody (scFvA7, Fitzerald) as described in Materials and methods . After incubation with VEGF (50 ng/ml for 10 min), in order to remove the antibody from the cell surface, cells were placed on ice and acid washed. In permeabilized and fixed HCAEC, VEGFR-2 was detected with an AlexaFluor488-conjugated secondary antibody and is shown in green. c-Src was labeled with AlexaFluor647-conjugated secondary antibody (red) and nuclei with DAPI (blue). (B) Bar graphs show image analysis for colocalization events using the NIH Image J plugin (as described in Materials and methods ). The graphs present the number of colocalization events normalized for the number of total VEGFR-2–positive immunofluorescence signals. Values are the mean of three experiments ± S.E.M., each containing numbers obtained from five random fields. * p

    Journal: PLoS ONE

    Article Title: Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

    doi: 10.1371/journal.pone.0028454

    Figure Lengend Snippet: VEGF induces subcellular co-localization of c-Src and internalized VEGFR-2 in an ROS-dependent manner. HCAEC transfected with control (Scram-si) (A) or si-p47 phox (B) were immunofluorescently double labeled for internalized VEGFR-2 (green) and c-Src (red). VEGFR-2 on HCAEC was labeled with single chain E-tagged antibody (scFvA7, Fitzerald) as described in Materials and methods . After incubation with VEGF (50 ng/ml for 10 min), in order to remove the antibody from the cell surface, cells were placed on ice and acid washed. In permeabilized and fixed HCAEC, VEGFR-2 was detected with an AlexaFluor488-conjugated secondary antibody and is shown in green. c-Src was labeled with AlexaFluor647-conjugated secondary antibody (red) and nuclei with DAPI (blue). (B) Bar graphs show image analysis for colocalization events using the NIH Image J plugin (as described in Materials and methods ). The graphs present the number of colocalization events normalized for the number of total VEGFR-2–positive immunofluorescence signals. Values are the mean of three experiments ± S.E.M., each containing numbers obtained from five random fields. * p

    Article Snippet: Colocalization of VEGFR-2 with c-Src or EEA1-positive endosome by immunofluorescence HCAEC were plated on fibronectin coated glass-bottom chamber slides (Lab-Tek II, Thermo Scientific) and starved overnight in 0.2% FBS containing EBM-2.

    Techniques: Transfection, Labeling, Incubation, Immunofluorescence

    Heterogeneous VEGF, VEGFR-1 and VEGFR-2 expression in colorectal cancer (CRC) endothelial cells (ECs): VEGF expression in ECs was heterogeneous with an labelling index (LI) range from 10.9 to 90% ( A , D ); Anti-VEGFR-1 immunostaining shows rare positivity with an LI range from nearly 0 ( B ) to 20% ( E ) in endothelial cells. VEGFR-2 shows a much wider immunostaining, with an LI range from 10 ( C ) to 72% ( F ). (Magnification: 400×). The arrows are pointed at vessels.

    Journal: International Journal of Molecular Sciences

    Article Title: The Prognostic Value of the Combination of Low VEGFR-1 and High VEGFR-2 Expression in Endothelial Cells of Colorectal Cancer

    doi: 10.3390/ijms19113536

    Figure Lengend Snippet: Heterogeneous VEGF, VEGFR-1 and VEGFR-2 expression in colorectal cancer (CRC) endothelial cells (ECs): VEGF expression in ECs was heterogeneous with an labelling index (LI) range from 10.9 to 90% ( A , D ); Anti-VEGFR-1 immunostaining shows rare positivity with an LI range from nearly 0 ( B ) to 20% ( E ) in endothelial cells. VEGFR-2 shows a much wider immunostaining, with an LI range from 10 ( C ) to 72% ( F ). (Magnification: 400×). The arrows are pointed at vessels.

    Article Snippet: Immunohistochemistry Standard immunohistochemistry (IHC) was applied as previously described [ , ] to serial 5-μm-thick sections to display VEGF, VEGFR-1 and VEGFR-2 expression using antibodies against VEGF (Thermo Scientific, Merelbeke, Belgium, polyclonal, dilution:1:50), VEGFR-1 (Abcam, Cambridge, UK, clone Y103, dilution 1:50) and VEGFR-2 (Cell Signaling, Danvers, MA, USA, clone 55B11, dilution 1:100).

    Techniques: Expressing, Immunostaining

    Quantitative image analysis on Tissue MicroArray (TMA): ( A ) After digitization of the TMA slides with a NanoZoomer Digital Slide Scanner (Hamamatsu, Hamamatsu City, Japan), blood vessels, highlighted by anti-CD34 immunostaining, were manually marked on the digital vascular endothelial growth factor receptor (VEGFR) slides by a pathologist using the NDP.view software. The tissue core diameter is 600 µm. ( B ) The delineated regions were imported into the Visiopharm software to quantify the VEGF, VEGFR-1 or VEGFR-2 expression levels using the labelling index (LI), i.e., the percentage of the immunoreactive area within the vessel area.

    Journal: International Journal of Molecular Sciences

    Article Title: The Prognostic Value of the Combination of Low VEGFR-1 and High VEGFR-2 Expression in Endothelial Cells of Colorectal Cancer

    doi: 10.3390/ijms19113536

    Figure Lengend Snippet: Quantitative image analysis on Tissue MicroArray (TMA): ( A ) After digitization of the TMA slides with a NanoZoomer Digital Slide Scanner (Hamamatsu, Hamamatsu City, Japan), blood vessels, highlighted by anti-CD34 immunostaining, were manually marked on the digital vascular endothelial growth factor receptor (VEGFR) slides by a pathologist using the NDP.view software. The tissue core diameter is 600 µm. ( B ) The delineated regions were imported into the Visiopharm software to quantify the VEGF, VEGFR-1 or VEGFR-2 expression levels using the labelling index (LI), i.e., the percentage of the immunoreactive area within the vessel area.

    Article Snippet: Immunohistochemistry Standard immunohistochemistry (IHC) was applied as previously described [ , ] to serial 5-μm-thick sections to display VEGF, VEGFR-1 and VEGFR-2 expression using antibodies against VEGF (Thermo Scientific, Merelbeke, Belgium, polyclonal, dilution:1:50), VEGFR-1 (Abcam, Cambridge, UK, clone Y103, dilution 1:50) and VEGFR-2 (Cell Signaling, Danvers, MA, USA, clone 55B11, dilution 1:100).

    Techniques: Microarray, Immunostaining, Software, Expressing