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    Name:
    Human VEGF Standard ELISA Development Kit
    Description:
    Human VEGF ELISA development kit contains the key components required for the quantitative measurement of natural and or recombinant human VEGF in a sandwich ELISA format Using the ELISA protocol the recommended microplates reagents and solutions the components supplied in this kit are sufficient to assay human VEGF approximately 1000 ELISA plate wells
    Catalog Number:
    900-k10
    Price:
    320.00
    Category:
    Detection Products
    Source:
    (BTI‐Tn‐5B1‐4) Hi‐5 Insect cells
    Quantity:
    Kit
    Buy from Supplier


    Structured Review

    PeproTech vegf a
    ANDV causes elevated <t>VEGF-A</t> levels in lung tissue models. (A) Levels of VEGF-A in supernatants. Data are presented as relative values of infected compared to uninfected models. (B) Levels of VEGF-A gene expression in models. Data were normalized to β-actin and presented as the change in induction relative to that of uninfected models. Data represent mean ± SEM of three independent experiments. In each experiment two infected and two uninfected models were analyzed. dpi; days post infection.
    Human VEGF ELISA development kit contains the key components required for the quantitative measurement of natural and or recombinant human VEGF in a sandwich ELISA format Using the ELISA protocol the recommended microplates reagents and solutions the components supplied in this kit are sufficient to assay human VEGF approximately 1000 ELISA plate wells
    https://www.bioz.com/result/vegf a/product/PeproTech
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vegf a - by Bioz Stars, 2021-03
    86/100 stars

    Images

    1) Product Images from "Andes Hantavirus-Infection of a 3D Human Lung Tissue Model Reveals a Late Peak in Progeny Virus Production Followed by Increased Levels of Proinflammatory Cytokines and VEGF-A"

    Article Title: Andes Hantavirus-Infection of a 3D Human Lung Tissue Model Reveals a Late Peak in Progeny Virus Production Followed by Increased Levels of Proinflammatory Cytokines and VEGF-A

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0149354

    ANDV causes elevated VEGF-A levels in lung tissue models. (A) Levels of VEGF-A in supernatants. Data are presented as relative values of infected compared to uninfected models. (B) Levels of VEGF-A gene expression in models. Data were normalized to β-actin and presented as the change in induction relative to that of uninfected models. Data represent mean ± SEM of three independent experiments. In each experiment two infected and two uninfected models were analyzed. dpi; days post infection.
    Figure Legend Snippet: ANDV causes elevated VEGF-A levels in lung tissue models. (A) Levels of VEGF-A in supernatants. Data are presented as relative values of infected compared to uninfected models. (B) Levels of VEGF-A gene expression in models. Data were normalized to β-actin and presented as the change in induction relative to that of uninfected models. Data represent mean ± SEM of three independent experiments. In each experiment two infected and two uninfected models were analyzed. dpi; days post infection.

    Techniques Used: Infection, Expressing

    2) Product Images from "Resistin enhances angiogenesis in osteosarcoma via the MAPK signaling pathway"

    Article Title: Resistin enhances angiogenesis in osteosarcoma via the MAPK signaling pathway

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.102423

    The MAPK signaling pathway is involved in resistin-promoted VEGF-A expression and contributes to angiogenesis. ( A ) Osteosarcoma 143B cells were incubated with resistin (10 ng/ml) for the indicated times, and ERK, JNK and p38 phosphorylation was determined by Western blot analysis. ( B ) Osteosarcoma 143B cells were pretreated with an ERKII inhibitor (10 μM), a JNK inhibitor (SP600125; 10 μM) and a p38 inhibitor (SB203580; 10 μM) for 30 min or transfected with ERK, JNK, and p38 siRNAs for 24 h, followed by resistin (10 ng/ml) stimulation for 24 h. VEGF-A expression was examined by qPCR. ( C – D ) Osteosarcoma 143B cells were pretreated with an ERKII inhibitor (10 μM), a JNK inhibitor (SP600125; 10 μM) and a p38 inhibitor (SB203580; 10 μM) for 30 min, or transfected with ERK, JNK and p38 siRNAs for 24 h, then stimulated with resistin (10 ng/ml) for 24 h. CM was collected and then applied to EPCs for 24 h. Cell migration and capillary-like structure formation in EPCs were examined by Transwell and tube formation assays, respectively. The results were obtained from three independent experiments. * p
    Figure Legend Snippet: The MAPK signaling pathway is involved in resistin-promoted VEGF-A expression and contributes to angiogenesis. ( A ) Osteosarcoma 143B cells were incubated with resistin (10 ng/ml) for the indicated times, and ERK, JNK and p38 phosphorylation was determined by Western blot analysis. ( B ) Osteosarcoma 143B cells were pretreated with an ERKII inhibitor (10 μM), a JNK inhibitor (SP600125; 10 μM) and a p38 inhibitor (SB203580; 10 μM) for 30 min or transfected with ERK, JNK, and p38 siRNAs for 24 h, followed by resistin (10 ng/ml) stimulation for 24 h. VEGF-A expression was examined by qPCR. ( C – D ) Osteosarcoma 143B cells were pretreated with an ERKII inhibitor (10 μM), a JNK inhibitor (SP600125; 10 μM) and a p38 inhibitor (SB203580; 10 μM) for 30 min, or transfected with ERK, JNK and p38 siRNAs for 24 h, then stimulated with resistin (10 ng/ml) for 24 h. CM was collected and then applied to EPCs for 24 h. Cell migration and capillary-like structure formation in EPCs were examined by Transwell and tube formation assays, respectively. The results were obtained from three independent experiments. * p

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

    Resistin mediates angiogenesis by inducing increases in VEGF-A expression in osteosarcoma cells. ( A – B ) After incubating osteosarcoma 143B cells with resistin (0–10 ng/ml) for 24 h, VEGF-A expression was measured by qPCR and ELISA. ( C – D ) Osteosarcoma 143B cells were pretreated with resistin (1–10 ng/ml) for 24 h. CM was collected and applied to EPCs for 24 h. Cell migration and capillary-like structure formation in EPCs were examined by Transwell and tube formation assays, respectively. The results were obtained from three independent experiments. * p
    Figure Legend Snippet: Resistin mediates angiogenesis by inducing increases in VEGF-A expression in osteosarcoma cells. ( A – B ) After incubating osteosarcoma 143B cells with resistin (0–10 ng/ml) for 24 h, VEGF-A expression was measured by qPCR and ELISA. ( C – D ) Osteosarcoma 143B cells were pretreated with resistin (1–10 ng/ml) for 24 h. CM was collected and applied to EPCs for 24 h. Cell migration and capillary-like structure formation in EPCs were examined by Transwell and tube formation assays, respectively. The results were obtained from three independent experiments. * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Migration

    Effects of resistin on VEGF-A-induced angiogenesis in the CAM model. ( A ) PBS, VEGF-A (50 ng/ml), control osteosarcoma 143B cell CM and resistin-treated osteosarcoma 143B CM were mixed in Matrigel and subjected to the CAM assay, then photographed with a stereomicroscope on developmental day 12. ( B ) Angiogenesis was quantified by counting the number of blood vessel branches. * p
    Figure Legend Snippet: Effects of resistin on VEGF-A-induced angiogenesis in the CAM model. ( A ) PBS, VEGF-A (50 ng/ml), control osteosarcoma 143B cell CM and resistin-treated osteosarcoma 143B CM were mixed in Matrigel and subjected to the CAM assay, then photographed with a stereomicroscope on developmental day 12. ( B ) Angiogenesis was quantified by counting the number of blood vessel branches. * p

    Techniques Used: Chick Chorioallantoic Membrane Assay

    Resistin promotes VEGF-A expression in osteosarcoma and contributes to angiogenesis through the NF-κB signaling pathway. ( A ) Osteosarcoma 143B cells were incubated with resistin (10 ng/ml) for the indicated times and p65 phosphorylation was determined by Western blot analysis. ( B ) Osteosarcoma 143B cells were pretreated with NF-κB inhibitors (PDTC 10 μM; TPCK 3 μM) for 30 min then stimulated with resistin (10 ng/ml) for 24 h. VEGF-A expression was examined by qPCR. ( C – D ) Osteosarcoma 143B cells were pretreated with NF-κB inhibitors (PDTC 10 μM; TPCK 3 μM) for 30 min then stimulated with resistin (10 ng/ml) for 24 h. CM was collected and applied to EPCs for 24 h. Cell migration and capillary-like structure formation in EPCs were examined by Transwell and tube formation assays, respectively. ( E ) Cells were transfected with indicated siRNA, the luciferase activity was examined. The results were obtained from three independent experiments. * p
    Figure Legend Snippet: Resistin promotes VEGF-A expression in osteosarcoma and contributes to angiogenesis through the NF-κB signaling pathway. ( A ) Osteosarcoma 143B cells were incubated with resistin (10 ng/ml) for the indicated times and p65 phosphorylation was determined by Western blot analysis. ( B ) Osteosarcoma 143B cells were pretreated with NF-κB inhibitors (PDTC 10 μM; TPCK 3 μM) for 30 min then stimulated with resistin (10 ng/ml) for 24 h. VEGF-A expression was examined by qPCR. ( C – D ) Osteosarcoma 143B cells were pretreated with NF-κB inhibitors (PDTC 10 μM; TPCK 3 μM) for 30 min then stimulated with resistin (10 ng/ml) for 24 h. CM was collected and applied to EPCs for 24 h. Cell migration and capillary-like structure formation in EPCs were examined by Transwell and tube formation assays, respectively. ( E ) Cells were transfected with indicated siRNA, the luciferase activity was examined. The results were obtained from three independent experiments. * p

    Techniques Used: Expressing, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Migration, Transfection, Luciferase, Activity Assay

    Clinical significance of resistin expression in tissue specimens from patients with osteosarcoma. ( A ) Tumor specimens were subjected to immunohistochemistry (IHC) evaluations with anti-resistin monoclonal antibody and the staining intensity was graded as 0 (no positive cell staining), 1 (1–24%, weakly positive), 2 (25–49%, moderately positive), or 3 (50–100%, strongly positive). ( B ) Positive correlations were observed between levels of resistin and VEGF-A expression.
    Figure Legend Snippet: Clinical significance of resistin expression in tissue specimens from patients with osteosarcoma. ( A ) Tumor specimens were subjected to immunohistochemistry (IHC) evaluations with anti-resistin monoclonal antibody and the staining intensity was graded as 0 (no positive cell staining), 1 (1–24%, weakly positive), 2 (25–49%, moderately positive), or 3 (50–100%, strongly positive). ( B ) Positive correlations were observed between levels of resistin and VEGF-A expression.

    Techniques Used: Expressing, Immunohistochemistry, Staining

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

    Article Title: Spatial regulation of VEGF receptor endocytosis in angiogenesis

    Journal: Nature cell biology

    doi: 10.1038/ncb2679

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

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

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

    Techniques Used: Two Tailed Test, Staining, Quantitation Assay

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

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

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

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

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

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

    4) Product Images from "Local Guidance of Emerging Vessel Sprouts Requires Soluble Flt-1 (VEGFR-1)"

    Article Title: Local Guidance of Emerging Vessel Sprouts Requires Soluble Flt-1 (VEGFR-1)

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2009.07.011

    Model for sFlt-1 regulation of vessel sprout guidance The schematic model illustrates how expression of sFlt-1 (blue; light blue “Y”s in E) in lateral base areas might regulate local sprout guidance by reducing the concentration of free VEGF (green; dark green diamonds in E) in regions adjacent to the emerging sprout (red). Dotted lines show potential endothelial cross-talk to reinforce heterogeneity. (A-D), changes with time, (E) blow-up of box in D showing how local sFlt-1 might neutralize VEGF-A (light blue diamonds) to provide a “corridor” for sprout emergence.
    Figure Legend Snippet: Model for sFlt-1 regulation of vessel sprout guidance The schematic model illustrates how expression of sFlt-1 (blue; light blue “Y”s in E) in lateral base areas might regulate local sprout guidance by reducing the concentration of free VEGF (green; dark green diamonds in E) in regions adjacent to the emerging sprout (red). Dotted lines show potential endothelial cross-talk to reinforce heterogeneity. (A-D), changes with time, (E) blow-up of box in D showing how local sFlt-1 might neutralize VEGF-A (light blue diamonds) to provide a “corridor” for sprout emergence.

    Techniques Used: Expressing, Concentration Assay

    Exogenous VEGF-A disrupts local guidance of sprouting retinal vessels (A-B) Confocal images of blood vessels from PBS- or VEGF-injected P5 mouse retinas stained with Alexa488-conjugated isolectin-B4. Arrowheads denote filopodia extended either in the direction of migration (A), or toward the sprout base (B). Scale Bar, 20 μm. (C-F) Vessel sprouts were analyzed for guidance parameters (C: * = p≤0.05; D: ** = p≤0.001; E and F: *** = p≤0.0001). (F) Values are averages + SD.
    Figure Legend Snippet: Exogenous VEGF-A disrupts local guidance of sprouting retinal vessels (A-B) Confocal images of blood vessels from PBS- or VEGF-injected P5 mouse retinas stained with Alexa488-conjugated isolectin-B4. Arrowheads denote filopodia extended either in the direction of migration (A), or toward the sprout base (B). Scale Bar, 20 μm. (C-F) Vessel sprouts were analyzed for guidance parameters (C: * = p≤0.05; D: ** = p≤0.001; E and F: *** = p≤0.0001). (F) Values are averages + SD.

    Techniques Used: Injection, Staining, Migration

    5) Product Images from "Dysregulated megakaryocyte distribution associated with nestin+ mesenchymal stem cells in immune thrombocytopenia"

    Article Title: Dysregulated megakaryocyte distribution associated with nestin+ mesenchymal stem cells in immune thrombocytopenia

    Journal: Blood Advances

    doi: 10.1182/bloodadvances.2018026690

    Platelet counts and MKs adjacent to sinusoidal vessels in mice treated with or without VEGF and/or AMD3100. (A) Platelet counts in the ITP model mice. Platelet counts were lower in the ITP model group than in the control group (66 ± 28 × 10 9 /L vs 117 ± 43 ×10 9 /L, respectively; P
    Figure Legend Snippet: Platelet counts and MKs adjacent to sinusoidal vessels in mice treated with or without VEGF and/or AMD3100. (A) Platelet counts in the ITP model mice. Platelet counts were lower in the ITP model group than in the control group (66 ± 28 × 10 9 /L vs 117 ± 43 ×10 9 /L, respectively; P

    Techniques Used: Mouse Assay

    CXCR4 and VEGFR1 mediated impaired MK distribution. (A) Concentration of MMP-9 in the BM analyzed by ELISA in the ITP group (n = 30) and control group (n = 28). (B) Concentration of VEGF in the BM analyzed by ELISA in the ITP group (n = 30) and control group (n = 28). (C) Expression of MMP-9 mRNA in CD41 + MKs analyzed by RT-PCR in the ITP group (n = 30) and control group (n = 28). (D) Expression of VEGF mRNA in CD41 + MKs analyzed by RT-PCR in the ITP group (n = 30) and control group (n = 28). (E) Expression of VEGFR1 mRNA in CD41 + MKs analyzed by RT-PCR in the ITP group (n = 30) and control group (n = 28). (F) VEGFR1 protein levels in CD41 + MKs analyzed by western blot analysis in the ITP group (n = 30) and control group (n = 28). (G) Model illustrating the BM vascular niche alterations, disrupted MK distribution, and reduced platelet production in ITP patients. Data are pooled from 10 independent experiments with 4 to 8 samples per experiment (A-E). Data are shown as mean ± SD. Each point represents the mean adjusted value of 3 replicates for each individual patient. P values were calculated using the Mann-Whitney U test. * P
    Figure Legend Snippet: CXCR4 and VEGFR1 mediated impaired MK distribution. (A) Concentration of MMP-9 in the BM analyzed by ELISA in the ITP group (n = 30) and control group (n = 28). (B) Concentration of VEGF in the BM analyzed by ELISA in the ITP group (n = 30) and control group (n = 28). (C) Expression of MMP-9 mRNA in CD41 + MKs analyzed by RT-PCR in the ITP group (n = 30) and control group (n = 28). (D) Expression of VEGF mRNA in CD41 + MKs analyzed by RT-PCR in the ITP group (n = 30) and control group (n = 28). (E) Expression of VEGFR1 mRNA in CD41 + MKs analyzed by RT-PCR in the ITP group (n = 30) and control group (n = 28). (F) VEGFR1 protein levels in CD41 + MKs analyzed by western blot analysis in the ITP group (n = 30) and control group (n = 28). (G) Model illustrating the BM vascular niche alterations, disrupted MK distribution, and reduced platelet production in ITP patients. Data are pooled from 10 independent experiments with 4 to 8 samples per experiment (A-E). Data are shown as mean ± SD. Each point represents the mean adjusted value of 3 replicates for each individual patient. P values were calculated using the Mann-Whitney U test. * P

    Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, MANN-WHITNEY

    6) Product Images from "Soluble vascular endothelial growth factor receptor 3 is essential for corneal alymphaticity"

    Article Title: Soluble vascular endothelial growth factor receptor 3 is essential for corneal alymphaticity

    Journal: Blood

    doi: 10.1182/blood-2012-08-453043

    sVEGFR-3 inhibits VEGF-C–induced VEGFR-2 phosphorylation but not VEGF-A–induced VEGFR-2 phosphorylation. (A) Immunoprecipitation by VEGFR-2 antibody from HUVEC lysate. The input shows soluble and membrane VEGFR-3 bands. However, VEGFR-3 bands were not detected after precipitation, indicating that the two receptors do not heterodimerize. (B) After 24 hours of serum starvation, HUVEC was stimulated with 20 ng/mL VEGF-A or 20 ng/mL VEGF-A + 500 ng/mL recombinant sVEGFR-3. Recombinant sVEGFR-3 did not block VEGF-A–induced VEGFR-2 phosphorylation. (C) After 24 hours of serum starvation, HUVEC was stimulated with 20 ng/mL, 100 ng/mL, or 500 ng/mL VEGF-C or 500 ng/mL VEGF-C + 1750 ng/mL recombinant sVEGFR-3. Recombinant sVEGFR-3 treatment inhibits VEGF-C–induced VEGFR-2 phosphorylation. IP, immunoprecipitation; WB, western blot.
    Figure Legend Snippet: sVEGFR-3 inhibits VEGF-C–induced VEGFR-2 phosphorylation but not VEGF-A–induced VEGFR-2 phosphorylation. (A) Immunoprecipitation by VEGFR-2 antibody from HUVEC lysate. The input shows soluble and membrane VEGFR-3 bands. However, VEGFR-3 bands were not detected after precipitation, indicating that the two receptors do not heterodimerize. (B) After 24 hours of serum starvation, HUVEC was stimulated with 20 ng/mL VEGF-A or 20 ng/mL VEGF-A + 500 ng/mL recombinant sVEGFR-3. Recombinant sVEGFR-3 did not block VEGF-A–induced VEGFR-2 phosphorylation. (C) After 24 hours of serum starvation, HUVEC was stimulated with 20 ng/mL, 100 ng/mL, or 500 ng/mL VEGF-C or 500 ng/mL VEGF-C + 1750 ng/mL recombinant sVEGFR-3. Recombinant sVEGFR-3 treatment inhibits VEGF-C–induced VEGFR-2 phosphorylation. IP, immunoprecipitation; WB, western blot.

    Techniques Used: Immunoprecipitation, Recombinant, Blocking Assay, Western Blot

    7) Product Images from "Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+ hemangiocytes"

    Article Title: Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+ hemangiocytes

    Journal: Nature medicine

    doi: 10.1038/nm1400

    Inhibition of CXCR4 and, to a lesser degree, of VEGFR1 blocks VEGF-A–induced mobilization of hemangiocytes and ischemic revascularization. ( a ) In the mouse ear pinna angiogenesis model, subcutaneous recombinant TPO (200 ng every 3 d) induced substantial formation and sprouting of neovessels similar to subcutaneous treatment with VEGF-A but with less edema formation (original magnification, ×10). PBS-injected ear pinna was used as a control. ( b ) Matrigel plugs with recombinant TPO showed substantial formation and branching of vascular channels comparable to VEGF-A (original magnification, ×400). Matrigel plugs loaded with PBS were used as controls. ( c ) Subcutaneous injection of low-dose recombinant TPO into ear pinna led to a twofold increase in plasma TPO and SDF-1 levels compared to PBS-treated controls ( n = 4 per group, * P
    Figure Legend Snippet: Inhibition of CXCR4 and, to a lesser degree, of VEGFR1 blocks VEGF-A–induced mobilization of hemangiocytes and ischemic revascularization. ( a ) In the mouse ear pinna angiogenesis model, subcutaneous recombinant TPO (200 ng every 3 d) induced substantial formation and sprouting of neovessels similar to subcutaneous treatment with VEGF-A but with less edema formation (original magnification, ×10). PBS-injected ear pinna was used as a control. ( b ) Matrigel plugs with recombinant TPO showed substantial formation and branching of vascular channels comparable to VEGF-A (original magnification, ×400). Matrigel plugs loaded with PBS were used as controls. ( c ) Subcutaneous injection of low-dose recombinant TPO into ear pinna led to a twofold increase in plasma TPO and SDF-1 levels compared to PBS-treated controls ( n = 4 per group, * P

    Techniques Used: Inhibition, Recombinant, Injection

    8) Product Images from "FLI1 and PKC co-activation promote highly efficient differentiation of human embryonic stem cells into endothelial-like cells"

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

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0162-9

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

    Techniques Used: Activation Assay, Over Expression, Expressing

    9) Product Images from "MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration"

    Article Title: MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600973

    Knockdown of MK2 suppresses VEGF-induced LIMK1 activation, stress fiber formation, and cell migration. ( A ) Knockdown of MK2 expression. MSS31 cells were transfected with the siRNA vector (mock), MK2 siRNA plasmid, or mutated MK2 siRNA plasmid. Expression of endogenous MK2 and LIMK1 was analyzed by immunoprecipitation and immunoblotting. ( B ) Suppression of LIMK1 activation. MSS31 cells transfected with siRNA plasmids were stimulated with VEGF for 15 min or cotransfected with HA-MKK6(DE). Endogenous LIMK1 was precipitated and subjected to an in vitro kinase assay. Relative kinase activities are shown in the bottom panel as means±s.d. of five experiments. ( C ). Bar, 50 μm. The bottom panel shows the data of quantitative analysis of triplicate experiments. ( D ) Suppression of cell migration. MSS31 cells transfected with YFP and siRNA plasmids were subjected to cell migration assay in the absence or presence of VEGF. Data are shown as means±s.d. of four experiments.
    Figure Legend Snippet: Knockdown of MK2 suppresses VEGF-induced LIMK1 activation, stress fiber formation, and cell migration. ( A ) Knockdown of MK2 expression. MSS31 cells were transfected with the siRNA vector (mock), MK2 siRNA plasmid, or mutated MK2 siRNA plasmid. Expression of endogenous MK2 and LIMK1 was analyzed by immunoprecipitation and immunoblotting. ( B ) Suppression of LIMK1 activation. MSS31 cells transfected with siRNA plasmids were stimulated with VEGF for 15 min or cotransfected with HA-MKK6(DE). Endogenous LIMK1 was precipitated and subjected to an in vitro kinase assay. Relative kinase activities are shown in the bottom panel as means±s.d. of five experiments. ( C ). Bar, 50 μm. The bottom panel shows the data of quantitative analysis of triplicate experiments. ( D ) Suppression of cell migration. MSS31 cells transfected with YFP and siRNA plasmids were subjected to cell migration assay in the absence or presence of VEGF. Data are shown as means±s.d. of four experiments.

    Techniques Used: Activation Assay, Migration, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, In Vitro, Kinase Assay, Cell Migration Assay

    Effects of expression of LIMK1 mutants on VEGF-induced tubule formation. ( A ) Bright-field micrographs showing the tubule formation of MSS31 cells expressing YFP, YFP-LIMK1(WT), or YFP-LIMK1(D460A) in Matrigel, in the presence or absence of VEGF. Bar, 1 mm. The tube-like structures were traced, as shown at the bottom of each micrograph, and the total tube length was quantified using the image software. The results are shown in the bottom panel, as means±s.d. of four experiments. ( B ) Phase contrast micrographs showing the tubule formation of MSS31 cells expressing LIMK1(T508V) or LIMK1(S310A/S323A/T508V) in Matrigel. Bar, 0.5 mm. The quantitative data of the total tube length are shown as means±s.d. of four experiments.
    Figure Legend Snippet: Effects of expression of LIMK1 mutants on VEGF-induced tubule formation. ( A ) Bright-field micrographs showing the tubule formation of MSS31 cells expressing YFP, YFP-LIMK1(WT), or YFP-LIMK1(D460A) in Matrigel, in the presence or absence of VEGF. Bar, 1 mm. The tube-like structures were traced, as shown at the bottom of each micrograph, and the total tube length was quantified using the image software. The results are shown in the bottom panel, as means±s.d. of four experiments. ( B ) Phase contrast micrographs showing the tubule formation of MSS31 cells expressing LIMK1(T508V) or LIMK1(S310A/S323A/T508V) in Matrigel. Bar, 0.5 mm. The quantitative data of the total tube length are shown as means±s.d. of four experiments.

    Techniques Used: Expressing, Software

    Kinase-negative LIMK1(D460A) suppresses VEGF-induced stress fiber formation. ( A ). Bar, 50 μm. ( B ) Quantitative analysis of data shown in (A). The percentages of the cells with thick stress fibers in the total CFP-positive cells are shown as means±s.d. of triplicate experiments.
    Figure Legend Snippet: Kinase-negative LIMK1(D460A) suppresses VEGF-induced stress fiber formation. ( A ). Bar, 50 μm. ( B ) Quantitative analysis of data shown in (A). The percentages of the cells with thick stress fibers in the total CFP-positive cells are shown as means±s.d. of triplicate experiments.

    Techniques Used:

    MK2 activates LIMK1 by phosphorylation of Ser-323. ( A ) MK2 phosphorylates and activates LIMK1. Myc-LIMK1(WT) and Myc-LIMK1(T508V) expressed in 293T cells were precipitated with anti-Myc antibody and incubated with [γ- 32 P]ATP and active GST-MK2-Myc. Reaction mixtures were separated by SDS–PAGE and analyzed by autoradiography. Relative values of 32 P incorporation into Myc-LIMK1 are indicated in the bottom left panel. After incubation with active MK2, Myc-LIMK1 immunoprecipitates were subjected to an in vitro kinase assay. Relative kinase activities are indicated in the bottom right panel. Data are means±s.d. of three independent experiments. ( B ) Alignment of sequences of putative MK2 phosphorylation sites in LIMK1 and HSP27, and the consensus sequence for MK2 substrates. An asterisk indicates the phosphorylation site. ( C ) MK2 activates LIMK1 by Ser-323 phosphorylation. Myc-LIMK1 mutants were expressed in 293T cells, immunoprecipitated, and incubated with [γ- 32 P]ATP and active GST-MK2-Myc, with or without active GST-p38. Both 32 P incorporation into Myc-LIMK1 and LIMK1 activity were analyzed as in (A). Relative values of 32 P incorporation and relative kinase activities are indicated in the bottom panels. ( D ) Activation of LIMK1 by Ser-323 phosphorylation in cultured cells. Myc-LIMK1 mutants were coexpressed with HA-MKK6(DE) plus Flag-p38 in 293T cells, immunoprecipitated, and subjected to an in vitro kinase assay. Relative kinase activities are indicated in the bottom panel as means±s.d. of five independent experiments. ( E ) Kinase activities of LIMK1 mutants. MSS31 cells transfected with Myc-LIMK1 mutants were stimulated with VEGF for 15 min. Myc-LIMK1 mutants were immunoprecipitated and subjected to an in vitro kinase assay. Relative kinase activities are shown in the bottom panel as means±s.d. of five experiments.
    Figure Legend Snippet: MK2 activates LIMK1 by phosphorylation of Ser-323. ( A ) MK2 phosphorylates and activates LIMK1. Myc-LIMK1(WT) and Myc-LIMK1(T508V) expressed in 293T cells were precipitated with anti-Myc antibody and incubated with [γ- 32 P]ATP and active GST-MK2-Myc. Reaction mixtures were separated by SDS–PAGE and analyzed by autoradiography. Relative values of 32 P incorporation into Myc-LIMK1 are indicated in the bottom left panel. After incubation with active MK2, Myc-LIMK1 immunoprecipitates were subjected to an in vitro kinase assay. Relative kinase activities are indicated in the bottom right panel. Data are means±s.d. of three independent experiments. ( B ) Alignment of sequences of putative MK2 phosphorylation sites in LIMK1 and HSP27, and the consensus sequence for MK2 substrates. An asterisk indicates the phosphorylation site. ( C ) MK2 activates LIMK1 by Ser-323 phosphorylation. Myc-LIMK1 mutants were expressed in 293T cells, immunoprecipitated, and incubated with [γ- 32 P]ATP and active GST-MK2-Myc, with or without active GST-p38. Both 32 P incorporation into Myc-LIMK1 and LIMK1 activity were analyzed as in (A). Relative values of 32 P incorporation and relative kinase activities are indicated in the bottom panels. ( D ) Activation of LIMK1 by Ser-323 phosphorylation in cultured cells. Myc-LIMK1 mutants were coexpressed with HA-MKK6(DE) plus Flag-p38 in 293T cells, immunoprecipitated, and subjected to an in vitro kinase assay. Relative kinase activities are indicated in the bottom panel as means±s.d. of five independent experiments. ( E ) Kinase activities of LIMK1 mutants. MSS31 cells transfected with Myc-LIMK1 mutants were stimulated with VEGF for 15 min. Myc-LIMK1 mutants were immunoprecipitated and subjected to an in vitro kinase assay. Relative kinase activities are shown in the bottom panel as means±s.d. of five experiments.

    Techniques Used: Incubation, SDS Page, Autoradiography, In Vitro, Kinase Assay, Sequencing, Immunoprecipitation, Activity Assay, Activation Assay, Cell Culture, Transfection

    VEGF induces LIMK1 activation and cofilin phosphorylation. ( A ) LIMK1 activation. HUVEC and MSS31 cells were stimulated with VEGF. At the indicated time, cells were lysed and LIMK1 was immunoprecipitated (IP) with anti-LIMK1, and subjected to an in vitro kinase assay. Reaction mixtures were analyzed by autoradiography ( 32 P-cofilin), Amido black staining (cofilin), and immunoblotting (IB) with anti-LIMK1 antibody. The bottom panel indicates the relative kinase activities of LIMK1, as measured by 32 P incorporation into cofilin. Data are means±s.d. of five independent experiments. ( B ) Cofilin phosphorylation. HUVEC and MSS31 cells were stimulated with VEGF. Cell lysates were analyzed by immunoblotting with anti-P-cofilin and anti-cofilin antibodies. The bottom panel indicates the relative P-cofilin levels as means±s.d. of five independent experiments. ( C ) Two-dimensional gel analyses of P-cofilin levels. MSS31 cells were stimulated with VEGF, and cell lysates were analyzed by two-dimensional gel electrophoresis, followed by immunoblotting with anti-cofilin antibody. The right panel indicates the mean abundance of P-cofilin (means±s.d. of triplicate experiments), as the percentage of total cofilin.
    Figure Legend Snippet: VEGF induces LIMK1 activation and cofilin phosphorylation. ( A ) LIMK1 activation. HUVEC and MSS31 cells were stimulated with VEGF. At the indicated time, cells were lysed and LIMK1 was immunoprecipitated (IP) with anti-LIMK1, and subjected to an in vitro kinase assay. Reaction mixtures were analyzed by autoradiography ( 32 P-cofilin), Amido black staining (cofilin), and immunoblotting (IB) with anti-LIMK1 antibody. The bottom panel indicates the relative kinase activities of LIMK1, as measured by 32 P incorporation into cofilin. Data are means±s.d. of five independent experiments. ( B ) Cofilin phosphorylation. HUVEC and MSS31 cells were stimulated with VEGF. Cell lysates were analyzed by immunoblotting with anti-P-cofilin and anti-cofilin antibodies. The bottom panel indicates the relative P-cofilin levels as means±s.d. of five independent experiments. ( C ) Two-dimensional gel analyses of P-cofilin levels. MSS31 cells were stimulated with VEGF, and cell lysates were analyzed by two-dimensional gel electrophoresis, followed by immunoblotting with anti-cofilin antibody. The right panel indicates the mean abundance of P-cofilin (means±s.d. of triplicate experiments), as the percentage of total cofilin.

    Techniques Used: Activation Assay, Immunoprecipitation, In Vitro, Kinase Assay, Autoradiography, Staining, Two-Dimensional Gel Electrophoresis, Electrophoresis

    A proposed signaling pathway for VEGF-induced LIMK1 activation and cofilin phosphorylation. VEGF induces activation of MK2 via the MKK6 and p38 MAPK signaling cascade. MK2 activates LIMK1 by phosphorylation of Ser-323, which in turn stimulates phosphorylation of cofilin. Together with MK2-mediated Hsp27 phosphorylation, cofilin phosphorylation stimulates stress fiber formation, cell migration, and tube formation.
    Figure Legend Snippet: A proposed signaling pathway for VEGF-induced LIMK1 activation and cofilin phosphorylation. VEGF induces activation of MK2 via the MKK6 and p38 MAPK signaling cascade. MK2 activates LIMK1 by phosphorylation of Ser-323, which in turn stimulates phosphorylation of cofilin. Together with MK2-mediated Hsp27 phosphorylation, cofilin phosphorylation stimulates stress fiber formation, cell migration, and tube formation.

    Techniques Used: Activation Assay, Migration

    Effects of expression of LIMK1 mutants on VEGF-induced cell migration and stress fiber formation. ( A ) Effects on cell migration. Migration of MSS31 cells expressing YFP alone (control) or YFP plus Myc-LIMK1 mutant was analyzed by Transwell assays. Relative numbers of migrating cells are shown as means±s.d. of four experiments. ( B ) Expression levels of LIMK1 mutants. MSS31 cells were transfected as above, and cell lysates were immunoprecipitated and immunoblotted with anti-LIMK1 antibody. ( C ). Bar, 50 μm. The right panel shows the data of quantitative analysis of triplicate experiments.
    Figure Legend Snippet: Effects of expression of LIMK1 mutants on VEGF-induced cell migration and stress fiber formation. ( A ) Effects on cell migration. Migration of MSS31 cells expressing YFP alone (control) or YFP plus Myc-LIMK1 mutant was analyzed by Transwell assays. Relative numbers of migrating cells are shown as means±s.d. of four experiments. ( B ) Expression levels of LIMK1 mutants. MSS31 cells were transfected as above, and cell lysates were immunoprecipitated and immunoblotted with anti-LIMK1 antibody. ( C ). Bar, 50 μm. The right panel shows the data of quantitative analysis of triplicate experiments.

    Techniques Used: Expressing, Migration, Mutagenesis, Transfection, Immunoprecipitation

    10) Product Images from "CCL3 promotes angiogenesis by dysregulation of miR-374b/ VEGF-A axis in human osteosarcoma cells"

    Article Title: CCL3 promotes angiogenesis by dysregulation of miR-374b/ VEGF-A axis in human osteosarcoma cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.6708

    CCL3 promotes VEGF-A expression and angiogenesis by downregulating miR-374b A. Cells were treated with CCL3 (1-10 ng/mL) for 24 h, and miR-374b expression was detected by RT-qPCR. B. Cells were transfected with wild-type (pmirGLO-VEGF-A-WT; WT) or mutant (pmirGLO-VEGF-A-MUT; MUT) 3′ UTR reporter assay plasmid for 24 h, and then incubated with CCL3 (1-10 ng/mL), the relative luciferase activity was measured. C. Cells were transfected with miR-374b mimic for 24 h, and then incubated with CCL3 (10 ng/mL), and the VEGF-A expression was detected by ELISA kit. D. The culture medium were collected as CM and then applied to EPCs for 24 h. The capillary-like structure formation in EPCs was examined by tube formation assay. E. Cells were pre-treated with SP600125, U0126, SB203580 for 30 min or pre-transferated with JNK-, ERK-, p38-, or multiple (combining JNK-, ERK-, and p38-) siRNA for 24 h, then treated cells with CCL3 (10 ng/mL) for 24 h. The miR-374b expression was detected by RT-qPCR. F. Mice were injected subcutaneously with Matrigel mixed with osteosarcoma CM for 7 days, and then the plugs were excised from mice, photographed and quantified the hemoglobin content. G H. Chick embryos were incubated with osteosarcoma CM or VEGF-A (0.5 and 50 ng/mL) for 4 days, and then photographed with a stereomicroscope. Each experiment was done in triplicate. Results are expressed as mean ± S.E.M. * P
    Figure Legend Snippet: CCL3 promotes VEGF-A expression and angiogenesis by downregulating miR-374b A. Cells were treated with CCL3 (1-10 ng/mL) for 24 h, and miR-374b expression was detected by RT-qPCR. B. Cells were transfected with wild-type (pmirGLO-VEGF-A-WT; WT) or mutant (pmirGLO-VEGF-A-MUT; MUT) 3′ UTR reporter assay plasmid for 24 h, and then incubated with CCL3 (1-10 ng/mL), the relative luciferase activity was measured. C. Cells were transfected with miR-374b mimic for 24 h, and then incubated with CCL3 (10 ng/mL), and the VEGF-A expression was detected by ELISA kit. D. The culture medium were collected as CM and then applied to EPCs for 24 h. The capillary-like structure formation in EPCs was examined by tube formation assay. E. Cells were pre-treated with SP600125, U0126, SB203580 for 30 min or pre-transferated with JNK-, ERK-, p38-, or multiple (combining JNK-, ERK-, and p38-) siRNA for 24 h, then treated cells with CCL3 (10 ng/mL) for 24 h. The miR-374b expression was detected by RT-qPCR. F. Mice were injected subcutaneously with Matrigel mixed with osteosarcoma CM for 7 days, and then the plugs were excised from mice, photographed and quantified the hemoglobin content. G H. Chick embryos were incubated with osteosarcoma CM or VEGF-A (0.5 and 50 ng/mL) for 4 days, and then photographed with a stereomicroscope. Each experiment was done in triplicate. Results are expressed as mean ± S.E.M. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Mutagenesis, Reporter Assay, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay, Tube Formation Assay, Mouse Assay, Injection

    Schematic presentation of the signaling pathways involved in CCL3-induced VEGF-A expression and angiogenesis of osteosarcoma cells CCL3 activates CCR5 receptor and JNK, ERK, or p38 pathway, then down-regulating miR-374b expression, which leads to VEGF-A expression and increases the angiogenesis of human osteosarcoma cells.
    Figure Legend Snippet: Schematic presentation of the signaling pathways involved in CCL3-induced VEGF-A expression and angiogenesis of osteosarcoma cells CCL3 activates CCR5 receptor and JNK, ERK, or p38 pathway, then down-regulating miR-374b expression, which leads to VEGF-A expression and increases the angiogenesis of human osteosarcoma cells.

    Techniques Used: Expressing

    Knockdown of CCL3 decreases VEGF-A expression and inhibits angiogenesis in human osteosarcoma cells A-D. The mRNA and protein expression of CCL3 and VEGF-A in Control- or CCL3-shRNA osteosarcoma cells were detected by RT-qPCR, western blot, and ELISA. E F. The culture medium were collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. G. Quantitative results of EPC tube formation assay. H. Chick embryos were incubated with osteosarcoma CM for 4 days, and then photographed with a stereomicroscope. Each experiment was done in triplicate. Results are expressed as mean ± S.E.M. * P
    Figure Legend Snippet: Knockdown of CCL3 decreases VEGF-A expression and inhibits angiogenesis in human osteosarcoma cells A-D. The mRNA and protein expression of CCL3 and VEGF-A in Control- or CCL3-shRNA osteosarcoma cells were detected by RT-qPCR, western blot, and ELISA. E F. The culture medium were collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. G. Quantitative results of EPC tube formation assay. H. Chick embryos were incubated with osteosarcoma CM for 4 days, and then photographed with a stereomicroscope. Each experiment was done in triplicate. Results are expressed as mean ± S.E.M. * P

    Techniques Used: Expressing, shRNA, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Migration, Tube Formation Assay, Incubation

    CCL3 promotes VEGF-A expression and angiogenesis through CCR5 receptor Cells were pretreated with CCR5 monoclonal antibody (mAb) (0-5 μg/ml) or the CCR5 antagonist (DAPTA) (0-1 nM) for 30 min, and then treated with CCL3 (10 ng/mL) for 24 h. The mRNA and protein expression of VEGF-A were detected by RT-qPCR A B. and ELISA C. D E. The MG-63 cells culture medium were collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. F. Quantitative results of EPCs tube formation assay. G. Chick embryos were incubated with osteosarcoma CM for 4 days, and then photographed with a stereomicroscope. Each experiment was done in triplicate. Results are expressed as mean ± S.E.M. * P
    Figure Legend Snippet: CCL3 promotes VEGF-A expression and angiogenesis through CCR5 receptor Cells were pretreated with CCR5 monoclonal antibody (mAb) (0-5 μg/ml) or the CCR5 antagonist (DAPTA) (0-1 nM) for 30 min, and then treated with CCL3 (10 ng/mL) for 24 h. The mRNA and protein expression of VEGF-A were detected by RT-qPCR A B. and ELISA C. D E. The MG-63 cells culture medium were collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. F. Quantitative results of EPCs tube formation assay. G. Chick embryos were incubated with osteosarcoma CM for 4 days, and then photographed with a stereomicroscope. Each experiment was done in triplicate. Results are expressed as mean ± S.E.M. * P

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

    JNK, p38, and ERK activation are involved in CCL3-promoted VEGF-A expression and contributing to angiogenesis Cells were pretreated with SP600125 (JNK inhibitor) (0-10 μM), U0126 (ERK inhibitor) (0-10 μM), or SB203580 (p38 inhibitor) (0-10 μM) for 30 min, and then treated CCL3 (10 ng/mL) for 24 h. The mRNA and protein expression of VEGF-A were detected by RT-qPCR A-C. and ELISA D. E F. The culture medium were collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. G. Cells were incubated with CCL3 (10 ng/mL) for the indicated times, and JNK, p38, and ERK phosphorylation were detected by western blot. The activation % was plotted phosphorylated target/total. Each experiment was done in triplicate. Results are expressed as mean ± S.E.M. * P
    Figure Legend Snippet: JNK, p38, and ERK activation are involved in CCL3-promoted VEGF-A expression and contributing to angiogenesis Cells were pretreated with SP600125 (JNK inhibitor) (0-10 μM), U0126 (ERK inhibitor) (0-10 μM), or SB203580 (p38 inhibitor) (0-10 μM) for 30 min, and then treated CCL3 (10 ng/mL) for 24 h. The mRNA and protein expression of VEGF-A were detected by RT-qPCR A-C. and ELISA D. E F. The culture medium were collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. G. Cells were incubated with CCL3 (10 ng/mL) for the indicated times, and JNK, p38, and ERK phosphorylation were detected by western blot. The activation % was plotted phosphorylated target/total. Each experiment was done in triplicate. Results are expressed as mean ± S.E.M. * P

    Techniques Used: Activation Assay, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Migration, Tube Formation Assay, Incubation, Western Blot

    CCL3 has importance effect on in vivo angiogenesis and clinical significance A. U-2 OS/Control-shRNA or U-2 OS/CCL3-shRNA cells were subcutaneously injected into the right flank, and tumors were monitored by bioluminescence imaging at 1 and 21 days. After 21 days, the mice were sacrificed and the tumors excised. The tumors were photographed with a microscope B. measured weight C. and volume D. and quantified the hemoglobin levels E. The mRNA expression of CCL3, VEGF-A and miR-374b in osteosarcoma patients was examined by RT-qPCR. The correlation between CCL3/VEGF-A F. CCL3/miR-374b G. and VEGF-A/miR-374b H. Each experiment was done in triplicate. Results are expressed as mean ± S.E.M. * P
    Figure Legend Snippet: CCL3 has importance effect on in vivo angiogenesis and clinical significance A. U-2 OS/Control-shRNA or U-2 OS/CCL3-shRNA cells were subcutaneously injected into the right flank, and tumors were monitored by bioluminescence imaging at 1 and 21 days. After 21 days, the mice were sacrificed and the tumors excised. The tumors were photographed with a microscope B. measured weight C. and volume D. and quantified the hemoglobin levels E. The mRNA expression of CCL3, VEGF-A and miR-374b in osteosarcoma patients was examined by RT-qPCR. The correlation between CCL3/VEGF-A F. CCL3/miR-374b G. and VEGF-A/miR-374b H. Each experiment was done in triplicate. Results are expressed as mean ± S.E.M. * P

    Techniques Used: In Vivo, shRNA, Injection, Imaging, Mouse Assay, Microscopy, Expressing, Quantitative RT-PCR

    CCL3 enhances angiogenesis by increasing VEGF-A expression in human osteosarcoma cells A. Thirty-one tumor specimens were immunostained (IHC) with anti-CCL3, CCR5 and VEGF-A antibody. B. Quantitative results and correlation between CCL3 and VEGF-A clinical grade. C D. Cells were treated with various concentration of CCL3, and the mRNA and protein expression were detected by RT-qPCR and ELISA. E F. Cells were pre-treated for 30 min with VEGF-A antibody (20 ng/mL), followed by stimulation with CCL3 (1-10 ng/mL) for 24 h. The culture medium was collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. G. Quantitative results of EPCs tube formation assay. Each experiment was done in triplicate. Results are expressed as mean ± S.E.M. * P
    Figure Legend Snippet: CCL3 enhances angiogenesis by increasing VEGF-A expression in human osteosarcoma cells A. Thirty-one tumor specimens were immunostained (IHC) with anti-CCL3, CCR5 and VEGF-A antibody. B. Quantitative results and correlation between CCL3 and VEGF-A clinical grade. C D. Cells were treated with various concentration of CCL3, and the mRNA and protein expression were detected by RT-qPCR and ELISA. E F. Cells were pre-treated for 30 min with VEGF-A antibody (20 ng/mL), followed by stimulation with CCL3 (1-10 ng/mL) for 24 h. The culture medium was collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. G. Quantitative results of EPCs tube formation assay. Each experiment was done in triplicate. Results are expressed as mean ± S.E.M. * P

    Techniques Used: Expressing, Immunohistochemistry, Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Migration, Tube Formation Assay

    11) Product Images from "Pharmacologically active microcarriers influence VEGF-A effects on mesenchymal stem cell survival"

    Article Title: Pharmacologically active microcarriers influence VEGF-A effects on mesenchymal stem cell survival

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2012.01662.x

    Cell growth in normoxia with and without factors (free-VEGF-A, FN-PAMs or FN-PAM-VEGF) after 3 and 6 days. Data are% variation with respect to mean value of MSCs kept under standard conditions for 3-days (MSC-3). After 3-days treatment there are not statistical differences among groups. After 6-days treatment cell growth was significantly increased by FN-PAM-VEGF-6 and even more by VEGF-6. * P
    Figure Legend Snippet: Cell growth in normoxia with and without factors (free-VEGF-A, FN-PAMs or FN-PAM-VEGF) after 3 and 6 days. Data are% variation with respect to mean value of MSCs kept under standard conditions for 3-days (MSC-3). After 3-days treatment there are not statistical differences among groups. After 6-days treatment cell growth was significantly increased by FN-PAM-VEGF-6 and even more by VEGF-6. * P

    Techniques Used:

    Representative blots and mean levels of the anti-apoptotic factor, Bcl-2 after 3-days treatment with active factors (free-VEGF-A and FN-PAM-VEGF). (A) MSC control group (B) MSC treated with FN-PAM-VEGF, and (C) MSC treated with free-VEGF-A. We normalized the expression of Bcl-2 for each condition to its matched loading control β actin and then where normalized with respect to the mean values of MSC-3. It can be appreciated an increase of Bcl-2 in FN-PAM-VEGF and a decrease in VEGF-A group. * P
    Figure Legend Snippet: Representative blots and mean levels of the anti-apoptotic factor, Bcl-2 after 3-days treatment with active factors (free-VEGF-A and FN-PAM-VEGF). (A) MSC control group (B) MSC treated with FN-PAM-VEGF, and (C) MSC treated with free-VEGF-A. We normalized the expression of Bcl-2 for each condition to its matched loading control β actin and then where normalized with respect to the mean values of MSC-3. It can be appreciated an increase of Bcl-2 in FN-PAM-VEGF and a decrease in VEGF-A group. * P

    Techniques Used: Expressing

    Western blot analysis of Akt (top panels), ERK1/2 (middle panels), and PKCε (bottom panels) after 3-days. ( A ) MSC control group ( B ) MSC treated with FN-PAM-VEGF, and ( C ) MSC treated with free-VEGF-A. The panels on the left are normalized phospho/total kinase ratios. The central panels are representative bands of total and phospho-kinases (some of the presented bands were not juxtaposed in the original film). The panels on the right show the normalized mean values of phospho-enzyme only. We normalized the expression of total kinases and phospho-kinases for each condition to its matched loading control β actin and then where normalized with respect to the mean values of MSCs kept under standard conditions for 3-days (MSC-3). It can be appreciated a reduced Akt phospho/total ratio in FN-PAM-VEGF, an increased ERK1/2 phospho/total ratio in VEGF-A, and an increased phospho/total ratio of PKCε in both FN-PAM-VEGF and VEGF-A groups. * P
    Figure Legend Snippet: Western blot analysis of Akt (top panels), ERK1/2 (middle panels), and PKCε (bottom panels) after 3-days. ( A ) MSC control group ( B ) MSC treated with FN-PAM-VEGF, and ( C ) MSC treated with free-VEGF-A. The panels on the left are normalized phospho/total kinase ratios. The central panels are representative bands of total and phospho-kinases (some of the presented bands were not juxtaposed in the original film). The panels on the right show the normalized mean values of phospho-enzyme only. We normalized the expression of total kinases and phospho-kinases for each condition to its matched loading control β actin and then where normalized with respect to the mean values of MSCs kept under standard conditions for 3-days (MSC-3). It can be appreciated a reduced Akt phospho/total ratio in FN-PAM-VEGF, an increased ERK1/2 phospho/total ratio in VEGF-A, and an increased phospho/total ratio of PKCε in both FN-PAM-VEGF and VEGF-A groups. * P

    Techniques Used: Western Blot, Expressing

    Cell growth in normoxia for 3-days (first four bars), and cell survival after 3-days hypoxia and 3-hrs reoxigenation (H/R), of MSC pre-treated or not with factors (free-VEGF-A, FN-PAMs or FN-PAM-VEGF) for 24-hrs. Data are% variation with respect to mean value of MSCs kept under standard conditions for 3-days (MSC-3-N). In normoxia only free-VEGF-A pre-treatment induced a significant increase in cell number. Only pre-treatment with FN-PAM-VEGF was able to counteract hypoxia-induced cell number reduction. * P
    Figure Legend Snippet: Cell growth in normoxia for 3-days (first four bars), and cell survival after 3-days hypoxia and 3-hrs reoxigenation (H/R), of MSC pre-treated or not with factors (free-VEGF-A, FN-PAMs or FN-PAM-VEGF) for 24-hrs. Data are% variation with respect to mean value of MSCs kept under standard conditions for 3-days (MSC-3-N). In normoxia only free-VEGF-A pre-treatment induced a significant increase in cell number. Only pre-treatment with FN-PAM-VEGF was able to counteract hypoxia-induced cell number reduction. * P

    Techniques Used:

    Western blot analysis of Akt (top panels), ERK1/2 (middle panels), and PKCε (bottom panels) after hypoxia/reoxygenation (H/R). ( A ) MSC control group ( B ) MSC treated with FN-PAM-VEGF, and ( C ) MSC treated with free-VEGF-A. The panels on the left are normalized phospho/total kinase ratios. The central panels are representative bands of total and phospho-kinases. The panels on the right show the normalized mean values of phospho-enzyme only. We normalized the expression of total kinases and phospho-kinases for each condition to its matched loading control β actin and then where normalized with respect to the mean values of MSCs subjected to H/R. It can be appreciated an increased Akt phospho/total ratio in both FN-PAM-VEGF and VEGF-A groups, an increased ERK1/2 phospho/total ratio in VEGF-A only, and an unchanged phospho/total ratio of PKCε in both FN-PAM-VEGF and VEGF-A groups. * P
    Figure Legend Snippet: Western blot analysis of Akt (top panels), ERK1/2 (middle panels), and PKCε (bottom panels) after hypoxia/reoxygenation (H/R). ( A ) MSC control group ( B ) MSC treated with FN-PAM-VEGF, and ( C ) MSC treated with free-VEGF-A. The panels on the left are normalized phospho/total kinase ratios. The central panels are representative bands of total and phospho-kinases. The panels on the right show the normalized mean values of phospho-enzyme only. We normalized the expression of total kinases and phospho-kinases for each condition to its matched loading control β actin and then where normalized with respect to the mean values of MSCs subjected to H/R. It can be appreciated an increased Akt phospho/total ratio in both FN-PAM-VEGF and VEGF-A groups, an increased ERK1/2 phospho/total ratio in VEGF-A only, and an unchanged phospho/total ratio of PKCε in both FN-PAM-VEGF and VEGF-A groups. * P

    Techniques Used: Western Blot, Expressing

    Western blot analysis of Akt (top panels), ERK1/2 (middle panels), and PKCε (bottom panels) after 1-day. ( A ) MSC control group ( B ) MSC treated with FN-PAM-VEGF, and ( C ) MSC treated with free-VEGF-A. The panels on the left are normalized phospho/total kinase ratios. The central panels are representative bands of total and phospho-kinases. The panels on the right show the normalized mean values of phospho-enzyme only. We normalized the expression of total kinases and phospho-kinases for each condition to its matched loading control β actin and then where normalized with respect to the mean values of MSCs kept under standard conditions for 1-day. Only FN-PAM-VEGF induces a significant reduction in phospho/total Akt ratio and phospho Akt level. * P
    Figure Legend Snippet: Western blot analysis of Akt (top panels), ERK1/2 (middle panels), and PKCε (bottom panels) after 1-day. ( A ) MSC control group ( B ) MSC treated with FN-PAM-VEGF, and ( C ) MSC treated with free-VEGF-A. The panels on the left are normalized phospho/total kinase ratios. The central panels are representative bands of total and phospho-kinases. The panels on the right show the normalized mean values of phospho-enzyme only. We normalized the expression of total kinases and phospho-kinases for each condition to its matched loading control β actin and then where normalized with respect to the mean values of MSCs kept under standard conditions for 1-day. Only FN-PAM-VEGF induces a significant reduction in phospho/total Akt ratio and phospho Akt level. * P

    Techniques Used: Western Blot, Expressing

    Western blot analysis of Akt (top panels), ERK1/2 (middle panels), and PKCε (bottom panels) after 30-min. ( A ) MSC control group ( B ) MSC treated with FN-PAM-VEGF, and ( C ) MSC treated with free-VEGF-A. The panels on the left are normalized phospho/total kinase ratios. The central panels are representative bands of total and phospho-kinases. The panels on the right show the normalized mean values of phospho-enzyme only. We normalized the expression of total kinases and phospho-kinases for each condition to its matched loading control β actin and then where normalized with respect to the mean values of MSCs kept under standard conditions for 30-min. After 30-min. treatment, only free-VEGF-A induces an increase in phosphorylation of Akt, ERK1/2 and PKCε. * P
    Figure Legend Snippet: Western blot analysis of Akt (top panels), ERK1/2 (middle panels), and PKCε (bottom panels) after 30-min. ( A ) MSC control group ( B ) MSC treated with FN-PAM-VEGF, and ( C ) MSC treated with free-VEGF-A. The panels on the left are normalized phospho/total kinase ratios. The central panels are representative bands of total and phospho-kinases. The panels on the right show the normalized mean values of phospho-enzyme only. We normalized the expression of total kinases and phospho-kinases for each condition to its matched loading control β actin and then where normalized with respect to the mean values of MSCs kept under standard conditions for 30-min. After 30-min. treatment, only free-VEGF-A induces an increase in phosphorylation of Akt, ERK1/2 and PKCε. * P

    Techniques Used: Western Blot, Expressing

    Illustrative release kinetics of VEGF-A from fibronectin-coated pharmacologically-active-microcarriers complexed with VEGF-A (FN-PAM-VEGF) and bioassay. The cumulative release of VEGF-A from FN-PAM up to 4 weeks was 21% that represent about 300 ng/ml of the entrapped protein. Each point represents the mean of triplicate experiments. The release profile was performed with 2.5 mg PAMs. VEGF-A collected from each sample of the release kinetics assay was able to stimulate HUVEC proliferation as an equal amount of native VEGF-A for a period of 7 days (data not shown).
    Figure Legend Snippet: Illustrative release kinetics of VEGF-A from fibronectin-coated pharmacologically-active-microcarriers complexed with VEGF-A (FN-PAM-VEGF) and bioassay. The cumulative release of VEGF-A from FN-PAM up to 4 weeks was 21% that represent about 300 ng/ml of the entrapped protein. Each point represents the mean of triplicate experiments. The release profile was performed with 2.5 mg PAMs. VEGF-A collected from each sample of the release kinetics assay was able to stimulate HUVEC proliferation as an equal amount of native VEGF-A for a period of 7 days (data not shown).

    Techniques Used:

    Representative blots and mean levels of the apoptotic factor, cleaved Caspase-3 after hypoxia/reoxygenation (H/R). (A) MSC control group (B) MSC treated with FN-PAM-VEGF and (C) MSC treated with free-VEGF-A. We normalized the expression of cleaved Caspase-3 for each condition to its matched loading control β actin and then where normalized with respect to the mean values of MSC. It can be appreciated a decrease of cleaved Caspase levels in FN-PAM-VEGF group only. * P
    Figure Legend Snippet: Representative blots and mean levels of the apoptotic factor, cleaved Caspase-3 after hypoxia/reoxygenation (H/R). (A) MSC control group (B) MSC treated with FN-PAM-VEGF and (C) MSC treated with free-VEGF-A. We normalized the expression of cleaved Caspase-3 for each condition to its matched loading control β actin and then where normalized with respect to the mean values of MSC. It can be appreciated a decrease of cleaved Caspase levels in FN-PAM-VEGF group only. * P

    Techniques Used: Expressing

    Time-lines and protocols for experimental groups in normoxia. Timing of various interventions is shown in relation to the onset of cell culture. Mesenchymal stem cells (MSCs) were kept under standard-normoxia conditions for 3- or 6-days (MSC-3 or MSC-6). Fibronectin-coated pharmacologically-active-microcarriers (FN-PAM), FN-PAM incorporating VEGF-A (FN-PAM-VEGF) or free VEGF-A were added at time 0, and after 3-days for the 6-days groups. Assessments were performed after 30-min. from the beginning of treatments and then 1-, 3- and 6-days after (upwards arrows). For other acronyms see also the text.
    Figure Legend Snippet: Time-lines and protocols for experimental groups in normoxia. Timing of various interventions is shown in relation to the onset of cell culture. Mesenchymal stem cells (MSCs) were kept under standard-normoxia conditions for 3- or 6-days (MSC-3 or MSC-6). Fibronectin-coated pharmacologically-active-microcarriers (FN-PAM), FN-PAM incorporating VEGF-A (FN-PAM-VEGF) or free VEGF-A were added at time 0, and after 3-days for the 6-days groups. Assessments were performed after 30-min. from the beginning of treatments and then 1-, 3- and 6-days after (upwards arrows). For other acronyms see also the text.

    Techniques Used: Cell Culture

    Time-lines and protocols for experimental groups in hypoxia/reoxygenation. Timing of various interventions is shown in relation to the onset of cell culture. Mesenchymal stem cells (MSCs) were kept under standard conditions for 1-day, then they were subjected to 3-days hypoxia and 3-hrs reoxygenation. Fibronectin-coated pharmacologically-active-microcarriers (FN-PAM), FN-PAM incorporating VEGF-A (FN-PAM-VEGF) or free VEGF-A were added at time 0. For comparative purpose we considered normoxic (-N) and hypoxic (-H) protocols. Assessments were performed after 30-min. from the beginning of treatments and then before and after hypoxia, and at the end of reoxygenation (upwards arrows). For other acronyms see also the text.
    Figure Legend Snippet: Time-lines and protocols for experimental groups in hypoxia/reoxygenation. Timing of various interventions is shown in relation to the onset of cell culture. Mesenchymal stem cells (MSCs) were kept under standard conditions for 1-day, then they were subjected to 3-days hypoxia and 3-hrs reoxygenation. Fibronectin-coated pharmacologically-active-microcarriers (FN-PAM), FN-PAM incorporating VEGF-A (FN-PAM-VEGF) or free VEGF-A were added at time 0. For comparative purpose we considered normoxic (-N) and hypoxic (-H) protocols. Assessments were performed after 30-min. from the beginning of treatments and then before and after hypoxia, and at the end of reoxygenation (upwards arrows). For other acronyms see also the text.

    Techniques Used: Cell Culture

    12) Product Images from "Preferential Attachment of Peritoneal Tumor Metastases to Omental Immune Aggregates and Possible Role of a Unique Vascular Microenvironment in Metastatic Survival and Growth"

    Article Title: Preferential Attachment of Peritoneal Tumor Metastases to Omental Immune Aggregates and Possible Role of a Unique Vascular Microenvironment in Metastatic Survival and Growth

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2006.051222

    Proposed model. As described, omental immune aggregates contain mesothelial cells, some of which are hypoxic (red) and therefore secrete VEGF-A ( black asterisks ). In a normal physiological setting (top), VEGF-A production can stimulate blood vessels to produce vascular sprouts (green outlined in red), resulting in the induction of angiogenesis and recovery of once-hypoxic mesothelial cells. The increase of vessels delivers more immune cells, along with additional oxygen and nutrients necessary to support the influx of cells. In a metastasis model (bottom), tumor cells invade aggregates and co-opt the existing dense vasculature. Because angiogenesis is already induced, new blood vessel formation is rapid, resulting in aggressive tumor growth.
    Figure Legend Snippet: Proposed model. As described, omental immune aggregates contain mesothelial cells, some of which are hypoxic (red) and therefore secrete VEGF-A ( black asterisks ). In a normal physiological setting (top), VEGF-A production can stimulate blood vessels to produce vascular sprouts (green outlined in red), resulting in the induction of angiogenesis and recovery of once-hypoxic mesothelial cells. The increase of vessels delivers more immune cells, along with additional oxygen and nutrients necessary to support the influx of cells. In a metastasis model (bottom), tumor cells invade aggregates and co-opt the existing dense vasculature. Because angiogenesis is already induced, new blood vessel formation is rapid, resulting in aggressive tumor growth.

    Techniques Used:

    VEGF-A production by cell types found within the omentum or peritoneal cavity. A: Cells (1 × 10 6 ) from the spleen, mesenteric LN, peritoneal lavage, or omentum were incubated in normoxic, hypoxic, or hypoxic plus brefeldin A (Golgi Plug, +GP) conditions for 16 hours, and VEGF-A in the supernatant was measured by ELISA. Subsets of cells from either the peritoneal cavity ( B ) or the omentum ( C ) were tested for VEGF-A production using intracellular staining and reported as the fold increase over controls as described in Materials and Methods. Cells not producing VEGF-A would have a fold increase of 1 (dotted line). Macrophages from the omentum were divided into those expressing low [Macs (L)] or high [Macs (H)] levels of F4/80. D: Phenotypic and morphological analysis of mesothelial cells isolated from single cell suspensions of the omentum. Gating on a population of VCAM + cells (left dot plot) revealed this to be CD45 − (middle dot plot). Further analysis of this VCAM + , CD45 − population demonstrated the cells were CD31 − (top histogram), and CD44 + (bottom histogram). The digital image is representative of these cells when grown in vitro after isolation by FACS (see below) showing a cobblestone morphology, which is typical of mesothelial cells. E: Mesothelial cells or CD45 + immune cells were sorted using flow cytometry and cultured in normoxic conditions, and VEGF-A production was measured after 16 hours of incubation as above. Each experiment described above was repeated at least three times with the exception of the sorted cells, which was done twice. Statistical significance was determined using a one-way analysis of variance followed by Bonferroni’s multiple comparison test.
    Figure Legend Snippet: VEGF-A production by cell types found within the omentum or peritoneal cavity. A: Cells (1 × 10 6 ) from the spleen, mesenteric LN, peritoneal lavage, or omentum were incubated in normoxic, hypoxic, or hypoxic plus brefeldin A (Golgi Plug, +GP) conditions for 16 hours, and VEGF-A in the supernatant was measured by ELISA. Subsets of cells from either the peritoneal cavity ( B ) or the omentum ( C ) were tested for VEGF-A production using intracellular staining and reported as the fold increase over controls as described in Materials and Methods. Cells not producing VEGF-A would have a fold increase of 1 (dotted line). Macrophages from the omentum were divided into those expressing low [Macs (L)] or high [Macs (H)] levels of F4/80. D: Phenotypic and morphological analysis of mesothelial cells isolated from single cell suspensions of the omentum. Gating on a population of VCAM + cells (left dot plot) revealed this to be CD45 − (middle dot plot). Further analysis of this VCAM + , CD45 − population demonstrated the cells were CD31 − (top histogram), and CD44 + (bottom histogram). The digital image is representative of these cells when grown in vitro after isolation by FACS (see below) showing a cobblestone morphology, which is typical of mesothelial cells. E: Mesothelial cells or CD45 + immune cells were sorted using flow cytometry and cultured in normoxic conditions, and VEGF-A production was measured after 16 hours of incubation as above. Each experiment described above was repeated at least three times with the exception of the sorted cells, which was done twice. Statistical significance was determined using a one-way analysis of variance followed by Bonferroni’s multiple comparison test.

    Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Magnetic Cell Separation, Isolation, In Vitro, FACS, Flow Cytometry, Cytometry, Cell Culture

    13) Product Images from "Matrix Metalloproteinase-1-mediated Up-regulation of Vascular Endothelial Growth Factor-2 in Endothelial Cells *"

    Article Title: Matrix Metalloproteinase-1-mediated Up-regulation of Vascular Endothelial Growth Factor-2 in Endothelial Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.417451

    MMP-1 stimulation augments VEGF-A-mediated signaling and endothelial proliferation. A, representative Western blot of phosphorylated signaling proteins (VEGFR2, ERK, JNK, and MAPK) from HUVECs treated overnight with or without MMP-1 followed by treatment with 10 ng/ml of VEGF-A for the indicated time points. B, densitometry measurements of p- VEGFR2 (after 5 min of VEGF-A stimulation) with ( black ) and without ( gray ) MMP-1 treatment for 24 h. Standardization is against levels of β-actin ( n = 6, *, p
    Figure Legend Snippet: MMP-1 stimulation augments VEGF-A-mediated signaling and endothelial proliferation. A, representative Western blot of phosphorylated signaling proteins (VEGFR2, ERK, JNK, and MAPK) from HUVECs treated overnight with or without MMP-1 followed by treatment with 10 ng/ml of VEGF-A for the indicated time points. B, densitometry measurements of p- VEGFR2 (after 5 min of VEGF-A stimulation) with ( black ) and without ( gray ) MMP-1 treatment for 24 h. Standardization is against levels of β-actin ( n = 6, *, p

    Techniques Used: Western Blot

    14) Product Images from "Acro-osteolysis is associated with enhanced osteoclastogenesis and higher blood VEGF levels in systemic sclerosis"

    Article Title: Acro-osteolysis is associated with enhanced osteoclastogenesis and higher blood VEGF levels in systemic sclerosis

    Journal: Arthritis & rheumatology (Hoboken, N.J.)

    doi: 10.1002/art.39424

    VEGF levels in SSc patients with AO and its association with OC formation
    Figure Legend Snippet: VEGF levels in SSc patients with AO and its association with OC formation

    Techniques Used:

    15) Product Images from "CXCR2 Mediates the Recruitment of Endothelial Progenitor Cells During Allergic Airways Remodeling"

    Article Title: CXCR2 Mediates the Recruitment of Endothelial Progenitor Cells During Allergic Airways Remodeling

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1002/stem.222

    Acute allergen challenge leads to increased levels of CXCR2 ligands and VEGF-A in lung homogenates. CXCL1, CXCL2, and VEGF-A levels in lung tissue homogenates were measured in samples from alum controls or samples from mice challenged with OVA at the
    Figure Legend Snippet: Acute allergen challenge leads to increased levels of CXCR2 ligands and VEGF-A in lung homogenates. CXCL1, CXCL2, and VEGF-A levels in lung tissue homogenates were measured in samples from alum controls or samples from mice challenged with OVA at the

    Techniques Used: Mouse Assay

    CXCL1 in association with CXCL2 is able to induce EPC recruitment to the lungs of sensitized mice. Mice were sensitized with alum or OVA (intraperitoneal) on days 0 and 12. On day 18 mice were challenged with CXCL1 in association with CXCL2, VEGF-A, or
    Figure Legend Snippet: CXCL1 in association with CXCL2 is able to induce EPC recruitment to the lungs of sensitized mice. Mice were sensitized with alum or OVA (intraperitoneal) on days 0 and 12. On day 18 mice were challenged with CXCL1 in association with CXCL2, VEGF-A, or

    Techniques Used: Mouse Assay

    16) Product Images from "Adiponectin promotes VEGF-A-dependent angiogenesis in human chondrosarcoma through PI3K, Akt, mTOR, and HIF-α pathway"

    Article Title: Adiponectin promotes VEGF-A-dependent angiogenesis in human chondrosarcoma through PI3K, Akt, mTOR, and HIF-α pathway

    Journal: Oncotarget

    doi:

    HIF-1α activation is involved in adiponectin-induced VEGF-A expression in human chondrosarcoma cells A. B. Cells were pretreated with the HIF-1 inhibitor (10 μM) for 30 min or transfected with HIF-1α siRNA for 24 h followed by stimulation with adiponectin (10 ng/ml) for 24 h, and VEGF-A expression was examined by qPCR, ELISA and western blotting. C. D. In addition, the medium was collected as CM and then applied to EPCs for 24 h. The capillary-like structures formation and cell migration in EPCs were examined by tube formation and Transwell assay. E. F. Cells were incubated with adiponectin (10 ng/ml) for the indicated times and HIF-1α expression was determined by western blotting and qPCR. G. Cells were pretreated for 30 min with ly294002, wortmannin, Akt inhibitor, rapamycin, or HIF-1 inhibitor for 30 min followed by stimulation with adiponectin (10 ng/ml) for 120 min. The HIF-1α activation was examined by chromatin immunoprecipitation and HRE luciferase activity. H. I. Cells were pretreated with ly294002, wortmannin, Akt inhibitor, rapamycin, or HIF-1 inhibitor for 30 min or transfected with siRNA of p85, Akt, mTOR, AdipoR1, AdipoR2, and HIF-1α before exposure to adiponectin. HRE luciferase activity was measured, and the results were normalized to β-galactosidase activity. Results are expressed as the mean ± S.E.M. *, p
    Figure Legend Snippet: HIF-1α activation is involved in adiponectin-induced VEGF-A expression in human chondrosarcoma cells A. B. Cells were pretreated with the HIF-1 inhibitor (10 μM) for 30 min or transfected with HIF-1α siRNA for 24 h followed by stimulation with adiponectin (10 ng/ml) for 24 h, and VEGF-A expression was examined by qPCR, ELISA and western blotting. C. D. In addition, the medium was collected as CM and then applied to EPCs for 24 h. The capillary-like structures formation and cell migration in EPCs were examined by tube formation and Transwell assay. E. F. Cells were incubated with adiponectin (10 ng/ml) for the indicated times and HIF-1α expression was determined by western blotting and qPCR. G. Cells were pretreated for 30 min with ly294002, wortmannin, Akt inhibitor, rapamycin, or HIF-1 inhibitor for 30 min followed by stimulation with adiponectin (10 ng/ml) for 120 min. The HIF-1α activation was examined by chromatin immunoprecipitation and HRE luciferase activity. H. I. Cells were pretreated with ly294002, wortmannin, Akt inhibitor, rapamycin, or HIF-1 inhibitor for 30 min or transfected with siRNA of p85, Akt, mTOR, AdipoR1, AdipoR2, and HIF-1α before exposure to adiponectin. HRE luciferase activity was measured, and the results were normalized to β-galactosidase activity. Results are expressed as the mean ± S.E.M. *, p

    Techniques Used: Activation Assay, Expressing, Transfection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Migration, Transwell Assay, Incubation, Chromatin Immunoprecipitation, Luciferase, Activity Assay

    Adiponectin promotes VEGF-A expression in human chondrosarcoma cells through AdipoR1/R2 receptor A. B. The JJ012 cells were incubated with adiponectin (1–10 ng/ml) for 24 h, and VEGF-A expression was examined by qPCR, ELISA, and western blotting. C. D. The JJ012 cells were pre-treated for 30 min with VEGF-A antibody (5 μg/ml) followed by stimulation with adiponectin (10 ng/ml) or incubated with adiponectin (1–10 ng/ml) for 24 h. The medium was collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. E. F. The JJ012 cells were transfected with AdipoR1 or AdipoR2 siRNA for 24 h followed by stimulation with adiponectin (10 ng/ml) for 24 h, and VEGF-A expression was examined by qPCR, ELISA and western blotting. G. H. In addition, the medium was collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. Results are expressed as the mean ± S.E.M. *, p
    Figure Legend Snippet: Adiponectin promotes VEGF-A expression in human chondrosarcoma cells through AdipoR1/R2 receptor A. B. The JJ012 cells were incubated with adiponectin (1–10 ng/ml) for 24 h, and VEGF-A expression was examined by qPCR, ELISA, and western blotting. C. D. The JJ012 cells were pre-treated for 30 min with VEGF-A antibody (5 μg/ml) followed by stimulation with adiponectin (10 ng/ml) or incubated with adiponectin (1–10 ng/ml) for 24 h. The medium was collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. E. F. The JJ012 cells were transfected with AdipoR1 or AdipoR2 siRNA for 24 h followed by stimulation with adiponectin (10 ng/ml) for 24 h, and VEGF-A expression was examined by qPCR, ELISA and western blotting. G. H. In addition, the medium was collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. Results are expressed as the mean ± S.E.M. *, p

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

    PI3K/Akt pathway is involved in adiponectin-increased VEGF-A expression A. B. Cells were pretreated with the ly294002 (10 μM), wortmannin (150 nM) and Akt inhibitor (10 μM) for 30 min or transfected with p85 and Akt siRNA for 24 h followed by stimulation with adiponectin (10 ng/ml) for 24 h, and VEGF-A expression was examined by qPCR, ELISA and western blotting. C. D. In addition, the medium was collected as CM and then applied to EPCs for 24 h. The capillary-like structures formation and cell migration in EPCs were examined by tube formation and Transwell assay. E. F. Cells were incubated with adiponectin (10 ng/ml) for the indicated times, or pretreated with the ly294002 for 30 min followed by stimulation with adiponectin for 60 min, and the Akt phosphorylation was determined by western blotting. Results are expressed as the mean ± S.E.M. *, p
    Figure Legend Snippet: PI3K/Akt pathway is involved in adiponectin-increased VEGF-A expression A. B. Cells were pretreated with the ly294002 (10 μM), wortmannin (150 nM) and Akt inhibitor (10 μM) for 30 min or transfected with p85 and Akt siRNA for 24 h followed by stimulation with adiponectin (10 ng/ml) for 24 h, and VEGF-A expression was examined by qPCR, ELISA and western blotting. C. D. In addition, the medium was collected as CM and then applied to EPCs for 24 h. The capillary-like structures formation and cell migration in EPCs were examined by tube formation and Transwell assay. E. F. Cells were incubated with adiponectin (10 ng/ml) for the indicated times, or pretreated with the ly294002 for 30 min followed by stimulation with adiponectin for 60 min, and the Akt phosphorylation was determined by western blotting. Results are expressed as the mean ± S.E.M. *, p

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

    PI3K/Akt-dependent mTOR signaling pathway is activated in response to adiponectin treatment of human chondrosarcoma cells A. B. Cells were pretreated with the rapamycin (30 nM) for 30 min or transfected with mTOR siRNA for 24 h followed by stimulation with adiponectin (10 ng/ml) for 24 h, and VEGF-A expression was examined by qPCR, ELISA and western blotting. C. D. In addition, the medium was collected as CM and then applied to EPCs for 24 h. The capillary-like structures formation and cell migration in EPCs were examined by tube formation and Transwell assay. E. F. Cells were incubated with adiponectin (10 ng/ml) for the indicated times, or pretreated with the ly294002 and Akt inhibitor for 30 min followed by stimulation with adiponectin for 60 min, and the mTOR phosphorylation was determined by western blotting. Results are expressed as the mean ± S.E.M. *, p
    Figure Legend Snippet: PI3K/Akt-dependent mTOR signaling pathway is activated in response to adiponectin treatment of human chondrosarcoma cells A. B. Cells were pretreated with the rapamycin (30 nM) for 30 min or transfected with mTOR siRNA for 24 h followed by stimulation with adiponectin (10 ng/ml) for 24 h, and VEGF-A expression was examined by qPCR, ELISA and western blotting. C. D. In addition, the medium was collected as CM and then applied to EPCs for 24 h. The capillary-like structures formation and cell migration in EPCs were examined by tube formation and Transwell assay. E. F. Cells were incubated with adiponectin (10 ng/ml) for the indicated times, or pretreated with the ly294002 and Akt inhibitor for 30 min followed by stimulation with adiponectin for 60 min, and the mTOR phosphorylation was determined by western blotting. Results are expressed as the mean ± S.E.M. *, p

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

    Knockdown adiponectin decreases VEGF-A expression in vitro and angiogenesis in vivo A. B. The protein and mRNA levels of adiponectin and VEGF-A in JJ012/control-shRNA and JJ012/adiponectin-shRNA cells were measured with the western blotting, qPCR, and ELISA. C. EPCs cells were incubated CM from JJ012/control-shRNA or JJ012/adiponectin-shRNA for 24 h, the cell migration was examined by Transwell, D. and the tube formation was photographed under microscope. E. Chick embryos were incubated 4 day with serum-free medium, JJ012/control-shRNA CM, or JJ012/adiponectin-shRNA CM, and then resected, fixed and photographed with a stereomicroscope. F. Mice were injected subcutaneously with Matrigel mixed with serum-free medium, JJ012/control-shRNA CM, or JJ012/adiponectin-shRNA CM for 7 days. The plugs were excised from mice and photographed or stained with CD31. Results are expressed as the mean ± S.E.M. *, p
    Figure Legend Snippet: Knockdown adiponectin decreases VEGF-A expression in vitro and angiogenesis in vivo A. B. The protein and mRNA levels of adiponectin and VEGF-A in JJ012/control-shRNA and JJ012/adiponectin-shRNA cells were measured with the western blotting, qPCR, and ELISA. C. EPCs cells were incubated CM from JJ012/control-shRNA or JJ012/adiponectin-shRNA for 24 h, the cell migration was examined by Transwell, D. and the tube formation was photographed under microscope. E. Chick embryos were incubated 4 day with serum-free medium, JJ012/control-shRNA CM, or JJ012/adiponectin-shRNA CM, and then resected, fixed and photographed with a stereomicroscope. F. Mice were injected subcutaneously with Matrigel mixed with serum-free medium, JJ012/control-shRNA CM, or JJ012/adiponectin-shRNA CM for 7 days. The plugs were excised from mice and photographed or stained with CD31. Results are expressed as the mean ± S.E.M. *, p

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

    The correlation of adiponectin, VEGF-A and tumor stages in human chondrosarcoma tissues A. Immunohistochemistry of adiponectin expression in healthy cartilage and chondrosarcoma tissue. Scale bar = 50 μm. B. Quantification of adiponectin staining. The correlation data are shown in C. Data represent the mean ± S.E.M.
    Figure Legend Snippet: The correlation of adiponectin, VEGF-A and tumor stages in human chondrosarcoma tissues A. Immunohistochemistry of adiponectin expression in healthy cartilage and chondrosarcoma tissue. Scale bar = 50 μm. B. Quantification of adiponectin staining. The correlation data are shown in C. Data represent the mean ± S.E.M.

    Techniques Used: Immunohistochemistry, Expressing, Staining

    17) Product Images from "Fat extract promotes angiogenesis in a murine model of limb ischemia: a novel cell-free therapeutic strategy"

    Article Title: Fat extract promotes angiogenesis in a murine model of limb ischemia: a novel cell-free therapeutic strategy

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-018-1014-y

    FE promotes endothelial cell proliferation, migration, and tube formation. a HUVECs treated with FE at indicated concentrations. Cell proliferation assessed using cell counting kit, and percentage of optical density values relative to control calculated. b HUVEC migration evaluated using cell migration assay. c Percentage of gap closure (24 h) quantified. d HUVECs added to solidified Matrigel in a serum-free medium in presence or absence of FE. VEGF 20 ng/ml used as positive control. After 6-h incubation, endothelial cell tube formation stained using Calcein-AM and assessed by fluorescence microscopy. e Assessment of number of branch points/mm 2 in each group. f Quantification of mean tube length. * p
    Figure Legend Snippet: FE promotes endothelial cell proliferation, migration, and tube formation. a HUVECs treated with FE at indicated concentrations. Cell proliferation assessed using cell counting kit, and percentage of optical density values relative to control calculated. b HUVEC migration evaluated using cell migration assay. c Percentage of gap closure (24 h) quantified. d HUVECs added to solidified Matrigel in a serum-free medium in presence or absence of FE. VEGF 20 ng/ml used as positive control. After 6-h incubation, endothelial cell tube formation stained using Calcein-AM and assessed by fluorescence microscopy. e Assessment of number of branch points/mm 2 in each group. f Quantification of mean tube length. * p

    Techniques Used: Migration, Cell Counting, Cell Migration Assay, Positive Control, Incubation, Staining, Fluorescence, Microscopy

    18) Product Images from "Dual Targeting of Endothelial and Cancer Cells Potentiates In Vitro Nanobody-Targeted Photodynamic Therapy"

    Article Title: Dual Targeting of Endothelial and Cancer Cells Potentiates In Vitro Nanobody-Targeted Photodynamic Therapy

    Journal: Cancers

    doi: 10.3390/cancers12102732

    Anti-VEGFR2 NBs blocked VEGF-induced proliferation and did not act as receptor agonists. ( a ) VEGF-A (50 nM) or NBs (1 µM) were added to the serum-starved MS1 cells and incubated for 15 min. VEGFR2 phosphorylation was measured in the total cell lysates by Western blotting: (top) staining of phosphorylated tyrosine 1175 of VEGFR2; (middle) staining of total VEGFR2; and (bottom) staining of actin. ( b ) Fold changes of P-VEGFR2 in MS1 cells treated with VEGF or nanobodies relative to the non-treated cells (NT). ( c ) MS1 cells treated with VEGF-A/NBs or both for 72 h followed by viability assay using AlamarBlue ® reagent. Data are presented as percent changes in cell proliferation relative to the non-treated cells (mean ± SD).
    Figure Legend Snippet: Anti-VEGFR2 NBs blocked VEGF-induced proliferation and did not act as receptor agonists. ( a ) VEGF-A (50 nM) or NBs (1 µM) were added to the serum-starved MS1 cells and incubated for 15 min. VEGFR2 phosphorylation was measured in the total cell lysates by Western blotting: (top) staining of phosphorylated tyrosine 1175 of VEGFR2; (middle) staining of total VEGFR2; and (bottom) staining of actin. ( b ) Fold changes of P-VEGFR2 in MS1 cells treated with VEGF or nanobodies relative to the non-treated cells (NT). ( c ) MS1 cells treated with VEGF-A/NBs or both for 72 h followed by viability assay using AlamarBlue ® reagent. Data are presented as percent changes in cell proliferation relative to the non-treated cells (mean ± SD).

    Techniques Used: Incubation, Western Blot, Staining, Viability Assay

    19) Product Images from "WISP-1 positively regulates angiogenesis by controlling VEGF-A expression in human osteosarcoma"

    Article Title: WISP-1 positively regulates angiogenesis by controlling VEGF-A expression in human osteosarcoma

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.421

    Involvement of HIF-1 α in WISP-1-induced VEGF-A expression and angiogenesis ( a and b ) MG-63 cells were pretreated with an HIF-1 inhibitor (HIF i; 10 μ M) for 30 min or HIF-1 α siRNA for 24 h, then treated with WISP-1 (30 ng/ml) for 24 h. mRNA and VEGF-A protein expression was detected by RT-qPCR and ELISA. Untreated cells were used as the control. ( c ) MG-63 cells were pretreated with an HIF i (10 μ M) for 30 min and then treated with WISP-1 (30 ng/ml) for 24 h. Culture medium was collected as CM and then applied to EPCs for 24 h. EPC capillary-like structure formation was examined by tube formation (bar=100 μ m). CM collected from untreated cells was used as the control. ( d ) MG-63 cells were incubated with WISP-1 (30 ng/ml) for the indicated times and HIF-1 α expression was detected by western blot (MW: molecular weight). ( e ) MG-63 cells were treated with WISP-1 (30 ng/ml) for 8 h, then incubated for 0–60 min with CHX 5 μ M. HIF-1 α protein expression was detected by western blot (MW: molecular weight) and the quantification results are shown in ( f ). ( g ) MG-63 cells were pretreated with HIF i or SP600125 for 30 min, then treated with WISP-1 (30 ng/ml) for 24 h. HIF-1 α protein expression was detected by western blot (MW, molecular weight). Untreated cells were used as the control. ( h ) MG-63 cells were pretreated with HIF i and SP600125 for 30 min or pre-transfected with FAK and JNK siRNA before exposure to WISP-1. HRE-luciferase activity was measured, and the results were normalized to β -galactosidase activity. Untreated cells were used as the control. Each experiment was performed in triplicate ( N =3). Results are expressed as the mean±S.E.M. * P
    Figure Legend Snippet: Involvement of HIF-1 α in WISP-1-induced VEGF-A expression and angiogenesis ( a and b ) MG-63 cells were pretreated with an HIF-1 inhibitor (HIF i; 10 μ M) for 30 min or HIF-1 α siRNA for 24 h, then treated with WISP-1 (30 ng/ml) for 24 h. mRNA and VEGF-A protein expression was detected by RT-qPCR and ELISA. Untreated cells were used as the control. ( c ) MG-63 cells were pretreated with an HIF i (10 μ M) for 30 min and then treated with WISP-1 (30 ng/ml) for 24 h. Culture medium was collected as CM and then applied to EPCs for 24 h. EPC capillary-like structure formation was examined by tube formation (bar=100 μ m). CM collected from untreated cells was used as the control. ( d ) MG-63 cells were incubated with WISP-1 (30 ng/ml) for the indicated times and HIF-1 α expression was detected by western blot (MW: molecular weight). ( e ) MG-63 cells were treated with WISP-1 (30 ng/ml) for 8 h, then incubated for 0–60 min with CHX 5 μ M. HIF-1 α protein expression was detected by western blot (MW: molecular weight) and the quantification results are shown in ( f ). ( g ) MG-63 cells were pretreated with HIF i or SP600125 for 30 min, then treated with WISP-1 (30 ng/ml) for 24 h. HIF-1 α protein expression was detected by western blot (MW, molecular weight). Untreated cells were used as the control. ( h ) MG-63 cells were pretreated with HIF i and SP600125 for 30 min or pre-transfected with FAK and JNK siRNA before exposure to WISP-1. HRE-luciferase activity was measured, and the results were normalized to β -galactosidase activity. Untreated cells were used as the control. Each experiment was performed in triplicate ( N =3). Results are expressed as the mean±S.E.M. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Molecular Weight, Transfection, Luciferase, Activity Assay

    WISP-1 promotes VEGF-A expression and angiogenesis by down-regulating miR-381 expression. ( a ) MG-63 cells were treated with WISP-1 (30 ng/ml) for 24 h, and miR-381 expression was detected by RT-qPCR. Cells treated with 0 mg/ml of WISP-1 were used as the control. ( b ) MG-63 cells (normal and stable) were transfected with 3′UTR reporter assay plasmids for 24 h, then the normal cells were incubated with WISP-1 (30 ng/ml) for 24 h. The relative luciferase activity was measured. Normal cells that were not treated with WISP-1 and the stable control-shRNA cells served as the controls. ( c ) MG-63 cells were transfected with miR-381 mimic for 24 h, then incubated with WISP-1 (30 ng/ml) and VEGF-A expression was detected by western blot (MW, molecular weight) and ELISA. Cells transfected with control miRNA were used as the control. ( d and e ) MG-63 cells were transfected with miR-381 mimic for 24 h, then incubated with WISP-1 (30 ng/ml). Culture medium was collected as CM and then applied to the EPCs for 24 h. Capillary-like structure formation and migration in the EPCs was examined by tube formation (bar=100 μ m) and the Transwell migration assay, respectively. CM collected from cells transfected with control miRNA was used as the control. ( f ) Cells were transfected with miR-381 mimic for 24 h, then incubated with WISP-1 (30 ng/ml). Culture medium was collected as CM. Chick embryos were incubated with CM for 4 days, then photographed with a stereomicroscope (bar=1 mm) and quantified by vessel count. CM collected from cells that were transfected with control miRNA were used as the control. ( g and h ) mRNA expression of WISP-1, VEGF-A, and miR-381 in human osteosarcoma tumor samples was examined by RT-qPCR to determine the correlation between miR-381/VEGF-A and miR-381/WISP-1. Each experiment was performed in triplicate ( N =3). Results are expressed as the mean±S.E.M. * P
    Figure Legend Snippet: WISP-1 promotes VEGF-A expression and angiogenesis by down-regulating miR-381 expression. ( a ) MG-63 cells were treated with WISP-1 (30 ng/ml) for 24 h, and miR-381 expression was detected by RT-qPCR. Cells treated with 0 mg/ml of WISP-1 were used as the control. ( b ) MG-63 cells (normal and stable) were transfected with 3′UTR reporter assay plasmids for 24 h, then the normal cells were incubated with WISP-1 (30 ng/ml) for 24 h. The relative luciferase activity was measured. Normal cells that were not treated with WISP-1 and the stable control-shRNA cells served as the controls. ( c ) MG-63 cells were transfected with miR-381 mimic for 24 h, then incubated with WISP-1 (30 ng/ml) and VEGF-A expression was detected by western blot (MW, molecular weight) and ELISA. Cells transfected with control miRNA were used as the control. ( d and e ) MG-63 cells were transfected with miR-381 mimic for 24 h, then incubated with WISP-1 (30 ng/ml). Culture medium was collected as CM and then applied to the EPCs for 24 h. Capillary-like structure formation and migration in the EPCs was examined by tube formation (bar=100 μ m) and the Transwell migration assay, respectively. CM collected from cells transfected with control miRNA was used as the control. ( f ) Cells were transfected with miR-381 mimic for 24 h, then incubated with WISP-1 (30 ng/ml). Culture medium was collected as CM. Chick embryos were incubated with CM for 4 days, then photographed with a stereomicroscope (bar=1 mm) and quantified by vessel count. CM collected from cells that were transfected with control miRNA were used as the control. ( g and h ) mRNA expression of WISP-1, VEGF-A, and miR-381 in human osteosarcoma tumor samples was examined by RT-qPCR to determine the correlation between miR-381/VEGF-A and miR-381/WISP-1. Each experiment was performed in triplicate ( N =3). Results are expressed as the mean±S.E.M. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Reporter Assay, Incubation, Luciferase, Activity Assay, shRNA, Western Blot, Molecular Weight, Enzyme-linked Immunosorbent Assay, Migration, Transwell Migration Assay

    WISP-1 increases VEGF-A expression and angiogenesis via α v β 3 integrin. ( a – c ) MG-63 cells were pretreated with RGD (10 μ M) or RAD (10 μ M) for 30 min, then treated with WISP-1 (30 ng/ml) for 24 h. mRNA and VEGF-A protein expression was detected by RT-qPCR, western blot (MW, molecular weight), and enzyme-linked immunosorbent assay. Untreated cells were used as the control. ( d and e ) MG-63 cells were pretreated with RGD (10 μ M) or RAD (10 μ M) for 30 min, then treated with WISP-1 (30 ng/ml) for 24 h. Culture medium was collected as CM and then applied to the EPCs for 24 h. EPC capillary-like structure formation was examined by tube formation (bar=100 μ m) and cell migration by the Transwell migration assay. CM collected from untreated cells was used as the control. Each experiment was performed in triplicate ( N =3). Results are expressed as the mean±S.E.M. * P
    Figure Legend Snippet: WISP-1 increases VEGF-A expression and angiogenesis via α v β 3 integrin. ( a – c ) MG-63 cells were pretreated with RGD (10 μ M) or RAD (10 μ M) for 30 min, then treated with WISP-1 (30 ng/ml) for 24 h. mRNA and VEGF-A protein expression was detected by RT-qPCR, western blot (MW, molecular weight), and enzyme-linked immunosorbent assay. Untreated cells were used as the control. ( d and e ) MG-63 cells were pretreated with RGD (10 μ M) or RAD (10 μ M) for 30 min, then treated with WISP-1 (30 ng/ml) for 24 h. Culture medium was collected as CM and then applied to the EPCs for 24 h. EPC capillary-like structure formation was examined by tube formation (bar=100 μ m) and cell migration by the Transwell migration assay. CM collected from untreated cells was used as the control. Each experiment was performed in triplicate ( N =3). Results are expressed as the mean±S.E.M. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Molecular Weight, Enzyme-linked Immunosorbent Assay, Migration, Transwell Migration Assay

    FAK and JNK signaling pathways are involved in WISP-1-induced VEGF-A expression and angiogenesis. ( a and b ) MG-63 cells were pretreated with a FAK inhibitor (FAK i; 10 μ M) for 30 min or a FAK siRNA for 24 h, before treatment with WISP-1 (30 ng/ml) for 24 h. mRNA was quantified using RT-qPCR and VEGF-A protein expression was assayed by enzyme-linked immunosorbent assay (ELISA). Untreated cells were used as the control. ( c and d ) MG-63 cells were pretreated with a JNK inhibitor (SP600125; 10 μ M) for 30 min or a JNK siRNA for 24 h, before treatment with WISP-1 (30 ng/ml) for 24 h. mRNA was quantified using RT-qPCR and VEGF-A protein expression was assayed by ELISA. Untreated cells were used as the control. ( e ) MG-63 cells were pretreated with a FAK i or SP600125 for 30 min, then treated with WISP-1 (30 ng/ml) for 24 h. Culture medium was collected as CM and then applied to EPCs for 24 h. EPC capillary-like structure formation was examined by tube formation (bar=100 μ m). CM collected from untreated cells was used as the control. ( f ) MG-63 cells were incubated with WISP-1 (30 ng/ml) for the indicated times; FAK and JNK phosphorylation was detected by western blot (MW, molecular weight). ( g ) Cells were pretreated for 30 min with FAK i or SP600125, followed by stimulation with WISP-1 (30 ng/ml). p-FAK and p-JNK expression were detected by western blot. Each experiment was performed in triplicate ( N =3). Results are expressed as the mean±S.E.M. * P
    Figure Legend Snippet: FAK and JNK signaling pathways are involved in WISP-1-induced VEGF-A expression and angiogenesis. ( a and b ) MG-63 cells were pretreated with a FAK inhibitor (FAK i; 10 μ M) for 30 min or a FAK siRNA for 24 h, before treatment with WISP-1 (30 ng/ml) for 24 h. mRNA was quantified using RT-qPCR and VEGF-A protein expression was assayed by enzyme-linked immunosorbent assay (ELISA). Untreated cells were used as the control. ( c and d ) MG-63 cells were pretreated with a JNK inhibitor (SP600125; 10 μ M) for 30 min or a JNK siRNA for 24 h, before treatment with WISP-1 (30 ng/ml) for 24 h. mRNA was quantified using RT-qPCR and VEGF-A protein expression was assayed by ELISA. Untreated cells were used as the control. ( e ) MG-63 cells were pretreated with a FAK i or SP600125 for 30 min, then treated with WISP-1 (30 ng/ml) for 24 h. Culture medium was collected as CM and then applied to EPCs for 24 h. EPC capillary-like structure formation was examined by tube formation (bar=100 μ m). CM collected from untreated cells was used as the control. ( f ) MG-63 cells were incubated with WISP-1 (30 ng/ml) for the indicated times; FAK and JNK phosphorylation was detected by western blot (MW, molecular weight). ( g ) Cells were pretreated for 30 min with FAK i or SP600125, followed by stimulation with WISP-1 (30 ng/ml). p-FAK and p-JNK expression were detected by western blot. Each experiment was performed in triplicate ( N =3). Results are expressed as the mean±S.E.M. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Molecular Weight

    WISP-1 promotes VEGF-A expression and angiogenesis. ( a and b ) Cultured medium was collected as CM and then applied to EPCs for 24 h. EPC capillary-like structure formation and cell migration were examined by tube formation (bar=100 μ m) and Transwell assay. Uncultured MG-63 medium was used as the control. ( c ) Quantitative results and an observed correlation between WISP-1 and VEGF-A in osteosarcoma patients ( N =20). ( d - f ) After MG-63 cells were treated with various concentrations of WISP-1, mRNA and protein expressions were detected by RT-qPCR, western blot (MW: molecular w eight), and enzyme-linked immunosorbent assay. Cells treated with 0 mg/ml of WISP-1 were used as the control. ( g and h ) MG-63 cells were treated with various concentrations of WISP-1. Culture medium was collected as CM and applied to the EPCs for 24 h. EPC capillary-like structure formation was examined by tube formation (bar=100 μ m) and cell migration by the Transwell migration assay. CM collected from cells treated with 0 mg/ml of WISP-1 was used as the control. Each experiment was performed in triplicate ( N =3). Results are expressed as the mean±S.E.M. * P
    Figure Legend Snippet: WISP-1 promotes VEGF-A expression and angiogenesis. ( a and b ) Cultured medium was collected as CM and then applied to EPCs for 24 h. EPC capillary-like structure formation and cell migration were examined by tube formation (bar=100 μ m) and Transwell assay. Uncultured MG-63 medium was used as the control. ( c ) Quantitative results and an observed correlation between WISP-1 and VEGF-A in osteosarcoma patients ( N =20). ( d - f ) After MG-63 cells were treated with various concentrations of WISP-1, mRNA and protein expressions were detected by RT-qPCR, western blot (MW: molecular w eight), and enzyme-linked immunosorbent assay. Cells treated with 0 mg/ml of WISP-1 were used as the control. ( g and h ) MG-63 cells were treated with various concentrations of WISP-1. Culture medium was collected as CM and applied to the EPCs for 24 h. EPC capillary-like structure formation was examined by tube formation (bar=100 μ m) and cell migration by the Transwell migration assay. CM collected from cells treated with 0 mg/ml of WISP-1 was used as the control. Each experiment was performed in triplicate ( N =3). Results are expressed as the mean±S.E.M. * P

    Techniques Used: Expressing, Cell Culture, Migration, Transwell Assay, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Transwell Migration Assay

    Knockdown of WISP-1 decreases angiogenesis in vivo . ( a ) Chick embryos were incubated with osteosarcoma 0% DMEM (control), osteosarcoma CM, or VEGF-A (50 ng/ml) for 4 days, then photographed with a stereomicroscope (bar=1 mm) and quantified by vessel count. ( b – d ) Mice were injected subcutaneously with Matrigel mixed with 0% DMEM (control) or osteosarcoma CM for 7 days, then the plugs were excised, photographed, stained with CD31 (bar=50 μm) and quantified for hemoglobin content. Each experiment was performed in triplicate ( N =3). Results are expressed as the mean±S.E.M. * P
    Figure Legend Snippet: Knockdown of WISP-1 decreases angiogenesis in vivo . ( a ) Chick embryos were incubated with osteosarcoma 0% DMEM (control), osteosarcoma CM, or VEGF-A (50 ng/ml) for 4 days, then photographed with a stereomicroscope (bar=1 mm) and quantified by vessel count. ( b – d ) Mice were injected subcutaneously with Matrigel mixed with 0% DMEM (control) or osteosarcoma CM for 7 days, then the plugs were excised, photographed, stained with CD31 (bar=50 μm) and quantified for hemoglobin content. Each experiment was performed in triplicate ( N =3). Results are expressed as the mean±S.E.M. * P

    Techniques Used: In Vivo, Incubation, Mouse Assay, Injection, Staining

    20) Product Images from "The Transcription Factor Bach1 Suppresses the Developmental Angiogenesis of Zebrafish"

    Article Title: The Transcription Factor Bach1 Suppresses the Developmental Angiogenesis of Zebrafish

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2017/2143875

    Bach1 occupies the TCF/LEF-binding site of the VEGF promoter and recruits HDAC1 to the VEGF promoter in HUVECs. (a) Potential TCF/LEF-binding site of the VEGF promoter is shown. Luciferase reporter constructs were created containing the truncated (−2650, −529, and −228) versions of the VEGF promoter; then the VEGF reporter or a pGL3-basic luciferase reporter and β -gal were cotransfected with a Bach1-coding vector or with an empty vector (Control) into HEK293T cells, and luciferase activity was evaluated 48 hours later ( n = 3; ∗∗ P
    Figure Legend Snippet: Bach1 occupies the TCF/LEF-binding site of the VEGF promoter and recruits HDAC1 to the VEGF promoter in HUVECs. (a) Potential TCF/LEF-binding site of the VEGF promoter is shown. Luciferase reporter constructs were created containing the truncated (−2650, −529, and −228) versions of the VEGF promoter; then the VEGF reporter or a pGL3-basic luciferase reporter and β -gal were cotransfected with a Bach1-coding vector or with an empty vector (Control) into HEK293T cells, and luciferase activity was evaluated 48 hours later ( n = 3; ∗∗ P

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

    Bach1 inhibits VEGF mRNA and protein expression in HUVECs. (a and b) mRNA or protein levels of VEGF 165 were compared in ConsiRNA- and Bach1siRNA-transfected HUVECs and in AdGFP and AdBach1-HUVECs. mRNA levels (a) were evaluated via quantitative PCR ( n = 4; ∗ P
    Figure Legend Snippet: Bach1 inhibits VEGF mRNA and protein expression in HUVECs. (a and b) mRNA or protein levels of VEGF 165 were compared in ConsiRNA- and Bach1siRNA-transfected HUVECs and in AdGFP and AdBach1-HUVECs. mRNA levels (a) were evaluated via quantitative PCR ( n = 4; ∗ P

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

    Bach1 suppresses endogenous Wnt/ β -catenin signaling and exogenous Wnt8a stimulated VEGF and IL-8 gene expression in zebrafish. (a) Representative fluorescent images of 36-hpf Tg(TOP:GFP) transgenic zebrafish embryos that had been injected with or without Bach1 mRNA. (b) The fluorescence intensity of GFP was quantified and expressed as the as the ratio of measurements Bach1 mRNA and Control group ( n = 12; ∗∗ P
    Figure Legend Snippet: Bach1 suppresses endogenous Wnt/ β -catenin signaling and exogenous Wnt8a stimulated VEGF and IL-8 gene expression in zebrafish. (a) Representative fluorescent images of 36-hpf Tg(TOP:GFP) transgenic zebrafish embryos that had been injected with or without Bach1 mRNA. (b) The fluorescence intensity of GFP was quantified and expressed as the as the ratio of measurements Bach1 mRNA and Control group ( n = 12; ∗∗ P

    Techniques Used: Expressing, Transgenic Assay, Injection, Fluorescence

    Exogenous administration of VEGF or IL-8 partially rescued Bach1-driven antiangiogenic response in HUVECs. (a) HUVECs were transfected with AdGFP or AdBach1 for 24 hours and then incubated with or without VEGF (50 ng/mL) or IL-8 (50 ng/mL) for 24 hours. Endothelial tube formation assay in Matrigel was performed in presence of VEGF or IL-8. Tube length was quantified and expressed as the fold change relative to AdGFP ( n = 4; ∗ P
    Figure Legend Snippet: Exogenous administration of VEGF or IL-8 partially rescued Bach1-driven antiangiogenic response in HUVECs. (a) HUVECs were transfected with AdGFP or AdBach1 for 24 hours and then incubated with or without VEGF (50 ng/mL) or IL-8 (50 ng/mL) for 24 hours. Endothelial tube formation assay in Matrigel was performed in presence of VEGF or IL-8. Tube length was quantified and expressed as the fold change relative to AdGFP ( n = 4; ∗ P

    Techniques Used: Transfection, Incubation, Endothelial Tube Formation Assay

    21) Product Images from "IL-1? Inhibits Human Osteoblast Migration"

    Article Title: IL-1? Inhibits Human Osteoblast Migration

    Journal: Molecular Medicine

    doi: 10.2119/molmed.2012.00058

    Influence of IL-1β on osteoblast migration toward several growth factors with IL-1β present in a uniform concentration (A) or as a gradient (B). In vitro migration was analyzed in a modified Boyden chamber using 8 μm polycarbonate filters. IL-1β was diluted in serum-free DMEM 100 pg/mL (black bars) and added either to both upper and lower (A) or only to lower (B) wells of the Boyden chamber as indicated. Serum-free DMEM was used as control (white bars). Growth factors were diluted in serum-free medium at the following concentrations: PDGF-BB (10 ng/mL), IGF-1 (100 ng/mL) and VEGF-A (100 ng/mL). Columns represent mean number of totally migrated cells, bars represent SEM from three independent donors. Significance was calculated with two-way ANOVA followed by a Bonferroni post hoc test; * P
    Figure Legend Snippet: Influence of IL-1β on osteoblast migration toward several growth factors with IL-1β present in a uniform concentration (A) or as a gradient (B). In vitro migration was analyzed in a modified Boyden chamber using 8 μm polycarbonate filters. IL-1β was diluted in serum-free DMEM 100 pg/mL (black bars) and added either to both upper and lower (A) or only to lower (B) wells of the Boyden chamber as indicated. Serum-free DMEM was used as control (white bars). Growth factors were diluted in serum-free medium at the following concentrations: PDGF-BB (10 ng/mL), IGF-1 (100 ng/mL) and VEGF-A (100 ng/mL). Columns represent mean number of totally migrated cells, bars represent SEM from three independent donors. Significance was calculated with two-way ANOVA followed by a Bonferroni post hoc test; * P

    Techniques Used: Migration, Concentration Assay, In Vitro, Modification

    22) Product Images from "Vascular Endothelial Growth Factor A Competitively Inhibits Platelet-Derived Growth Factor (PDGF)-Dependent Activation of PDGF Receptor and Subsequent Signaling Events and Cellular Responses"

    Article Title: Vascular Endothelial Growth Factor A Competitively Inhibits Platelet-Derived Growth Factor (PDGF)-Dependent Activation of PDGF Receptor and Subsequent Signaling Events and Cellular Responses

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.06668-11

    VEGF-A bound to PDGFRα and prevented PDGF-dependent dimerization and internalization. (a) VEGF-A competes with PDGF-B in a dose-dependent manner for binding to PDGFRα. Fα cells were grown in 24-well plates until nearly confluent,
    Figure Legend Snippet: VEGF-A bound to PDGFRα and prevented PDGF-dependent dimerization and internalization. (a) VEGF-A competes with PDGF-B in a dose-dependent manner for binding to PDGFRα. Fα cells were grown in 24-well plates until nearly confluent,

    Techniques Used: Binding Assay

    VEGF-A antagonized PDGFRα and PDGF-driven cellular response without engaging VEGFRs. (a) Expression of VEGFR1 and VEGFR2 in MEFs, PAE/KDR, and ARPE-19α cells. MEFs, PAE/KDR, and ARPE-19α cells were cultured in 10% serum to near
    Figure Legend Snippet: VEGF-A antagonized PDGFRα and PDGF-driven cellular response without engaging VEGFRs. (a) Expression of VEGFR1 and VEGFR2 in MEFs, PAE/KDR, and ARPE-19α cells. MEFs, PAE/KDR, and ARPE-19α cells were cultured in 10% serum to near

    Techniques Used: Expressing, Cell Culture

    VEGF-A was present in PVR vitreous and inhibited PDGF-dependent activation of PDGFRα. (a) Ribbon structural alignments of human PDGF-B (blue) and human VEGF-A (yellow) crystal structures. Monomers (left) superimpose with a root mean square (RMS)
    Figure Legend Snippet: VEGF-A was present in PVR vitreous and inhibited PDGF-dependent activation of PDGFRα. (a) Ribbon structural alignments of human PDGF-B (blue) and human VEGF-A (yellow) crystal structures. Monomers (left) superimpose with a root mean square (RMS)

    Techniques Used: Activation Assay

    Neutralizing VEGF-A enabled vitreal PDGFs to activate PDGFRα and inhibit its indirect activation by vitreous. (a) Anti-VEGF-A promoted vitreal PDGFs to activate PDGFRα. Serum-starved ARPE-19α cells were treated 10 min at 37°C
    Figure Legend Snippet: Neutralizing VEGF-A enabled vitreal PDGFs to activate PDGFRα and inhibit its indirect activation by vitreous. (a) Anti-VEGF-A promoted vitreal PDGFs to activate PDGFRα. Serum-starved ARPE-19α cells were treated 10 min at 37°C

    Techniques Used: Activation Assay

    23) Product Images from "Paracrine Signals From Liver Sinusoidal Endothelium Regulate Hepatitis C Virus Replication"

    Article Title: Paracrine Signals From Liver Sinusoidal Endothelium Regulate Hepatitis C Virus Replication

    Journal: Hepatology (Baltimore, Md.)

    doi: 10.1002/hep.26571

    Reduced endothelial VEGFR-2 signaling in chronic liver disease. Representative image of VEGFR-2 expression in normal human liver showing a predominant sinusoidal staining pattern (A). VEGF-A expression in normal and diseased liver samples (n = 6) was quantified by qRT-PCR (B). Liver biopsy protein lysates (30 μg) from normal, HCV-infected, or alcohol-related liver disease (each n = 6, representative blots from three donors shown) were probed for proteins of interest as indicated (C). Densitometry was used to quantify VE-cadherin (D) and phosphorylated VEGFR-2 expression, allowing us to determine their relative expression in all samples (E). Statistical comparisons were made with the Kruskal-Wallis test with Dunn's correction where * P
    Figure Legend Snippet: Reduced endothelial VEGFR-2 signaling in chronic liver disease. Representative image of VEGFR-2 expression in normal human liver showing a predominant sinusoidal staining pattern (A). VEGF-A expression in normal and diseased liver samples (n = 6) was quantified by qRT-PCR (B). Liver biopsy protein lysates (30 μg) from normal, HCV-infected, or alcohol-related liver disease (each n = 6, representative blots from three donors shown) were probed for proteins of interest as indicated (C). Densitometry was used to quantify VE-cadherin (D) and phosphorylated VEGFR-2 expression, allowing us to determine their relative expression in all samples (E). Statistical comparisons were made with the Kruskal-Wallis test with Dunn's correction where * P

    Techniques Used: Expressing, Staining, Quantitative RT-PCR, Infection

    LSEC secreted factors increase hepatocellular HCV replication. Conditioned media (CM) was collected from LSEC seeded at 4 × 10 4 /cm 2 for 24 hours in the absence of VEGF-A and was diluted 1:2 with fresh media and used to treat Huh-7.5 (A), or PHH (B) for 18 hours prior to infecting with HCV JFH-1. CM was replenished following infection. HCV infection was enumerated by quantifying NS5A expressing cells or HCV RNA levels as indicated and the data expressed relative to mock-treated cells. In replicate experiments with Huh-7.5, the number of cells following mock or CM treatment was quantified after 48 hours (C). Huh-7.5 supporting a JFH-1 subgenomic replicon were treated with CM for 48 hours and HCV RNA quantified (D). Data are mean ±1 SD of n = 4 donor LSEC CM. Statistical comparisons were made with the Mann-Whitney U test where * P
    Figure Legend Snippet: LSEC secreted factors increase hepatocellular HCV replication. Conditioned media (CM) was collected from LSEC seeded at 4 × 10 4 /cm 2 for 24 hours in the absence of VEGF-A and was diluted 1:2 with fresh media and used to treat Huh-7.5 (A), or PHH (B) for 18 hours prior to infecting with HCV JFH-1. CM was replenished following infection. HCV infection was enumerated by quantifying NS5A expressing cells or HCV RNA levels as indicated and the data expressed relative to mock-treated cells. In replicate experiments with Huh-7.5, the number of cells following mock or CM treatment was quantified after 48 hours (C). Huh-7.5 supporting a JFH-1 subgenomic replicon were treated with CM for 48 hours and HCV RNA quantified (D). Data are mean ±1 SD of n = 4 donor LSEC CM. Statistical comparisons were made with the Mann-Whitney U test where * P

    Techniques Used: Infection, Expressing, MANN-WHITNEY

    p38 MAPK activation negatively regulates BMP4 expression. LSEC were pretreated with control solvent or kinase inhibitors (all 10 μM) for 1 hour, washed, and incubated with VEGF-A (10 ng/mL) as indicated. Conditioned media (CM) was collected and used to treat Huh-7.5 cells for 18 hours prior to infecting with HCV JFH-1 (A). LSEC were incubated with anti-VEGFR neutralizing antibodies for 1 hour prior to treating with VEGF ligands for 10 minutes before lysing for immunoblotting (B). LSEC were incubated with increasing concentrations of SB203580, or solvent control, before washing and treating with VEGF-A (10 ng/mL) for 8 hours. Cellular BMP4 levels were measured by qRT-PCR (C) or CM collected and used to treat Huh-7.5 cells for 18 hours prior to infecting with HCV JFH-1 (D). Data are mean ±1 SD of n = 4 donor LSEC. Statistical comparisons were made with the Kruskal-Wallis test with Dunn's correction where * P
    Figure Legend Snippet: p38 MAPK activation negatively regulates BMP4 expression. LSEC were pretreated with control solvent or kinase inhibitors (all 10 μM) for 1 hour, washed, and incubated with VEGF-A (10 ng/mL) as indicated. Conditioned media (CM) was collected and used to treat Huh-7.5 cells for 18 hours prior to infecting with HCV JFH-1 (A). LSEC were incubated with anti-VEGFR neutralizing antibodies for 1 hour prior to treating with VEGF ligands for 10 minutes before lysing for immunoblotting (B). LSEC were incubated with increasing concentrations of SB203580, or solvent control, before washing and treating with VEGF-A (10 ng/mL) for 8 hours. Cellular BMP4 levels were measured by qRT-PCR (C) or CM collected and used to treat Huh-7.5 cells for 18 hours prior to infecting with HCV JFH-1 (D). Data are mean ±1 SD of n = 4 donor LSEC. Statistical comparisons were made with the Kruskal-Wallis test with Dunn's correction where * P

    Techniques Used: Activation Assay, Expressing, Incubation, Quantitative RT-PCR

    Endothelial BMP4 expression is regulated by way of VEGFR-2. LSEC were starved of VEGF overnight and stimulated with VEGF-A, PlGF, or VEGF-E (all 10 ng/mL) for 8 hours, lysed, and RNA prepared for qRT-PCR analysis of BMP4 mRNA (A). Conditioned media (CM) from LSEC treated with the above ligands was collected and used to treat Huh-7.5 cells for 18 hours prior to infecting with HCV JFH-1 (B). Data are mean ±1 SD of n = 4 donor LSEC. Statistical comparisons were made with the Kruskal-Wallis test with Dunn's correction as appropriate and where ** P
    Figure Legend Snippet: Endothelial BMP4 expression is regulated by way of VEGFR-2. LSEC were starved of VEGF overnight and stimulated with VEGF-A, PlGF, or VEGF-E (all 10 ng/mL) for 8 hours, lysed, and RNA prepared for qRT-PCR analysis of BMP4 mRNA (A). Conditioned media (CM) from LSEC treated with the above ligands was collected and used to treat Huh-7.5 cells for 18 hours prior to infecting with HCV JFH-1 (B). Data are mean ±1 SD of n = 4 donor LSEC. Statistical comparisons were made with the Kruskal-Wallis test with Dunn's correction as appropriate and where ** P

    Techniques Used: Expressing, Quantitative RT-PCR

    BMP4 is negatively regulated by VEGF and stimulates HCV replication. LSEC were starved of VEGF overnight and stimulated with VEGF-A as indicated for 8 hours and lysed to quantify BMP4 mRNA (A) or protein (B). Conditioned media (CM) from untreated or VEGF-A treated LSEC were incubated with a neutralizing anti-BMP4 antibody (10 μg/mL) and screened for its effect on Huh-7.5 permissivity (C). Huh-7.5 were treated with recombinant BMP4 for 48 hours and the cells assessed for their proliferative capacity (D) and permissivity to support HCV JFH-1 infection. Infection was enumerated by quantifying NS5A-expressing cells (E) or HCV RNA (F). Primary hepatocytes were treated with BMP4 (100 ng/mL) for 18 hours, inoculated with HCV JFH-1, and infection assessed by measuring HCV RNA (G). Data are mean ±1 SD of n = 4 donor LSEC. Statistical comparisons were made with the Kruskal-Wallis test with Dunn's correction (A,C,E,F), or the Mann-Whitney U test (G) and where * P
    Figure Legend Snippet: BMP4 is negatively regulated by VEGF and stimulates HCV replication. LSEC were starved of VEGF overnight and stimulated with VEGF-A as indicated for 8 hours and lysed to quantify BMP4 mRNA (A) or protein (B). Conditioned media (CM) from untreated or VEGF-A treated LSEC were incubated with a neutralizing anti-BMP4 antibody (10 μg/mL) and screened for its effect on Huh-7.5 permissivity (C). Huh-7.5 were treated with recombinant BMP4 for 48 hours and the cells assessed for their proliferative capacity (D) and permissivity to support HCV JFH-1 infection. Infection was enumerated by quantifying NS5A-expressing cells (E) or HCV RNA (F). Primary hepatocytes were treated with BMP4 (100 ng/mL) for 18 hours, inoculated with HCV JFH-1, and infection assessed by measuring HCV RNA (G). Data are mean ±1 SD of n = 4 donor LSEC. Statistical comparisons were made with the Kruskal-Wallis test with Dunn's correction (A,C,E,F), or the Mann-Whitney U test (G) and where * P

    Techniques Used: Incubation, Recombinant, Infection, Expressing, MANN-WHITNEY

    Paracrine VEGF signaling reduces HCV infection of LSEC-hepatocyte co-cultures. CM was collected from LSEC-treated with increasing concentrations of recombinant VEGF-A and screened for its effect on Huh-7.5 permissivity to support HCV JFH-1 infection. Infection was enumerated by quantifying NS5A-expressing cells and the data expressed relative to mock control (black) (A). Data are mean ±1 SD of n = 4 donor LSEC CM. Statistical comparisons were made with the Kruskal-Wallis test with Dunn's correction where ** P
    Figure Legend Snippet: Paracrine VEGF signaling reduces HCV infection of LSEC-hepatocyte co-cultures. CM was collected from LSEC-treated with increasing concentrations of recombinant VEGF-A and screened for its effect on Huh-7.5 permissivity to support HCV JFH-1 infection. Infection was enumerated by quantifying NS5A-expressing cells and the data expressed relative to mock control (black) (A). Data are mean ±1 SD of n = 4 donor LSEC CM. Statistical comparisons were made with the Kruskal-Wallis test with Dunn's correction where ** P

    Techniques Used: Infection, Recombinant, Expressing

    24) Product Images from "Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA"

    Article Title: Robust differentiation of human pluripotent stem cells into endothelial cells via temporal modulation of ETV2 with modified mRNA

    Journal: bioRxiv

    doi: 10.1101/2020.03.02.973289

    VEGF signaling and activation of endogenous ETV2 in the standard S1-S2 differentiation protocol. ( A-D ) Generation of h-iPSCs-KDR -/- and h-iPSCs-ETV2 -/- clones by CRISPR/Cas9. ( A ) Sanger sequencing of the two edited alleles encoding the 3 rd exon of KDR . ( B ) Flow cytometry showed the conversion of h-iPSCs-KDR -/- into FLK1-/CD31+ h-iECs at 48 h using the early modETV2 protocol. ( C ) Sanger sequencing of the two edited alleles encoding the 4 th exon of ETV2 . ( D ) Immunofluorescence staining for ETV2 at 72 h using the S1-S2 differentiation protocol. Nuclei stained by DAPI. Scale bar, 200 μm. ( E ) Differences in differentiation efficiency between four alternative S1-S2 methodologies and the S1-modETV2 protocol for h-iPSC clones lacking either ETV2 and KDR (h-iPSC-ETV2 -/- and h-iPSC-KDR -/- ). Only S1-modETV2 protocol could successfully derive h-iECs from either h-iPSC-ETV2 -/- or h-iPSC-KDR -/- cell lines with high efficiency. In contrast, the four alternative S1-S2 methodologies failed to get any h-iECs. ( F-G ) Effect of VEGF-A concentration on h-iEC yield using the S1-S2 differentiation protocol. ( F ) Dose dependent conversion efficiency of h-iPSCs into CD31+ h-iECs by flow cytometry. ( G ) Immunofluorescence staining for ETV2 and VE-Cadherin at 72 h with different concentrations of VEGF-A. Nuclei stained by DAPI. Scale bar, 50 μm.
    Figure Legend Snippet: VEGF signaling and activation of endogenous ETV2 in the standard S1-S2 differentiation protocol. ( A-D ) Generation of h-iPSCs-KDR -/- and h-iPSCs-ETV2 -/- clones by CRISPR/Cas9. ( A ) Sanger sequencing of the two edited alleles encoding the 3 rd exon of KDR . ( B ) Flow cytometry showed the conversion of h-iPSCs-KDR -/- into FLK1-/CD31+ h-iECs at 48 h using the early modETV2 protocol. ( C ) Sanger sequencing of the two edited alleles encoding the 4 th exon of ETV2 . ( D ) Immunofluorescence staining for ETV2 at 72 h using the S1-S2 differentiation protocol. Nuclei stained by DAPI. Scale bar, 200 μm. ( E ) Differences in differentiation efficiency between four alternative S1-S2 methodologies and the S1-modETV2 protocol for h-iPSC clones lacking either ETV2 and KDR (h-iPSC-ETV2 -/- and h-iPSC-KDR -/- ). Only S1-modETV2 protocol could successfully derive h-iECs from either h-iPSC-ETV2 -/- or h-iPSC-KDR -/- cell lines with high efficiency. In contrast, the four alternative S1-S2 methodologies failed to get any h-iECs. ( F-G ) Effect of VEGF-A concentration on h-iEC yield using the S1-S2 differentiation protocol. ( F ) Dose dependent conversion efficiency of h-iPSCs into CD31+ h-iECs by flow cytometry. ( G ) Immunofluorescence staining for ETV2 and VE-Cadherin at 72 h with different concentrations of VEGF-A. Nuclei stained by DAPI. Scale bar, 50 μm.

    Techniques Used: Activation Assay, Clone Assay, CRISPR, Sequencing, Flow Cytometry, Immunofluorescence, Staining, Concentration Assay

    Inefficient activation of endogenous ETV2 in intermediate h-MPCs during the standard S1-S2 differentiation protocol. ( A ) Time course analysis of mRNA expression (qRT– PCR) of transcription factors TBXT (mesodermal commitment) and ETV2 (endothelial commitment) during the standard S1-S2 differentiation protocol. Relative fold change normalized to GAPDH expression. ( B ) Immunofluorescence staining for Brachyury in h-iPSCs at 48 h during the S1-S2 protocol. h-iPSCs lacking endogenous ETV2 (h-iPSCs-ETV2 -/- ) served as control. Nuclei stained by DAPI. Scale bar, 100 μm. Percentage of Brachyury+ cells at day 1. ( C ) Immunofluorescence staining for ETV2 in h-iPSCs at 72 h during the S1-S2 protocol. h-iPSCs-ETV2 -/- served as control. Nuclei stained by DAPI. Scale bar, 100 μm. Percentage of ETV2+ cells at day 3. ( D ) Effect of VEGF-A concentration on the percentages of ETV2+ cells at 72 h and CD31+ cells at 96 h during the S1-S2 protocol measured by immunofluorescence staining (ETV2) and flow cytometry (CD31). ( E ) Immunofluorescence staining for ETV2 in h-iPSCs during the optimized S1-modETV2 protocol. h-iPSCs-ETV2 -/- served as control. Nuclei stained by DAPI. Scale bar, 100 μm. Percentage of ETV2+ cells after transfection with modRNA. ( F ) Conversion efficiency of h-iPSCs into CD31+ h-iECs by flow cytometry. Comparison of the standard S1-S2 and the S1-modETV2 protocols. h-iPSCs-ETV2 -/- , h-iPSCs-KDR -/- , and h-iPSCs treated with the VEGFR2 inhibitor SU5416 served as controls. In panels b, c, and e, bars represent mean ± s.d.; n = 4; n.s. = no statistical differences and *** P
    Figure Legend Snippet: Inefficient activation of endogenous ETV2 in intermediate h-MPCs during the standard S1-S2 differentiation protocol. ( A ) Time course analysis of mRNA expression (qRT– PCR) of transcription factors TBXT (mesodermal commitment) and ETV2 (endothelial commitment) during the standard S1-S2 differentiation protocol. Relative fold change normalized to GAPDH expression. ( B ) Immunofluorescence staining for Brachyury in h-iPSCs at 48 h during the S1-S2 protocol. h-iPSCs lacking endogenous ETV2 (h-iPSCs-ETV2 -/- ) served as control. Nuclei stained by DAPI. Scale bar, 100 μm. Percentage of Brachyury+ cells at day 1. ( C ) Immunofluorescence staining for ETV2 in h-iPSCs at 72 h during the S1-S2 protocol. h-iPSCs-ETV2 -/- served as control. Nuclei stained by DAPI. Scale bar, 100 μm. Percentage of ETV2+ cells at day 3. ( D ) Effect of VEGF-A concentration on the percentages of ETV2+ cells at 72 h and CD31+ cells at 96 h during the S1-S2 protocol measured by immunofluorescence staining (ETV2) and flow cytometry (CD31). ( E ) Immunofluorescence staining for ETV2 in h-iPSCs during the optimized S1-modETV2 protocol. h-iPSCs-ETV2 -/- served as control. Nuclei stained by DAPI. Scale bar, 100 μm. Percentage of ETV2+ cells after transfection with modRNA. ( F ) Conversion efficiency of h-iPSCs into CD31+ h-iECs by flow cytometry. Comparison of the standard S1-S2 and the S1-modETV2 protocols. h-iPSCs-ETV2 -/- , h-iPSCs-KDR -/- , and h-iPSCs treated with the VEGFR2 inhibitor SU5416 served as controls. In panels b, c, and e, bars represent mean ± s.d.; n = 4; n.s. = no statistical differences and *** P

    Techniques Used: Activation Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Concentration Assay, Flow Cytometry, Transfection

    25) Product Images from "Attachment of Flexible Heparin Chains to Gelatin Scaffolds Improves Endothelial Cell Infiltration"

    Article Title: Attachment of Flexible Heparin Chains to Gelatin Scaffolds Improves Endothelial Cell Infiltration

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2011.0712

    Immunofluorescence staining of sections from grafts explanted on day 7. Images shown are representative of unmodified scaffolds (S), heparinized scaffolds (SH), and heparinized scaffolds incubated with VEGF-A and FGF-2 (SHVF), respectively. Sections were
    Figure Legend Snippet: Immunofluorescence staining of sections from grafts explanted on day 7. Images shown are representative of unmodified scaffolds (S), heparinized scaffolds (SH), and heparinized scaffolds incubated with VEGF-A and FGF-2 (SHVF), respectively. Sections were

    Techniques Used: Immunofluorescence, Staining, Incubation

    Infiltration of granulocytes and activated phagocytes into scaffolds in vivo. (A) Infiltration of granulocytes (Gr-1, red) into grafts of unmodified scaffolds (S), heparinized scaffolds (SH), and heparinized scaffolds incubated with VEGF-A and FGF-2 (SHVF)
    Figure Legend Snippet: Infiltration of granulocytes and activated phagocytes into scaffolds in vivo. (A) Infiltration of granulocytes (Gr-1, red) into grafts of unmodified scaffolds (S), heparinized scaffolds (SH), and heparinized scaffolds incubated with VEGF-A and FGF-2 (SHVF)

    Techniques Used: In Vivo, Incubation

    Quantification of cell infiltration in explanted unmodified scaffolds (S, ♦ ), heparinized scaffolds (SH, ■), and heparinized scaffolds incubated with VEGF-A and FGF-2 (SHVF, ▲), explanted after days 1, 3, or 7. Specific infiltration
    Figure Legend Snippet: Quantification of cell infiltration in explanted unmodified scaffolds (S, ♦ ), heparinized scaffolds (SH, ■), and heparinized scaffolds incubated with VEGF-A and FGF-2 (SHVF, ▲), explanted after days 1, 3, or 7. Specific infiltration

    Techniques Used: Incubation

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    Article Title: Immunoglobulin E induces VEGF production in mast cells and potentiates their pro-tumorigenic actions through a Fyn kinase-dependent mechanism
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    Article Title: High-mobility group box 1 protein is implicated in advanced glycation end products-induced vascular endothelial growth factor A production in the rat retinal ganglion cell line RGC-5
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    Expressing:

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    Quantitation Assay:

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    PeproTech vegf
    (a) <t>VEGF</t> and (b) <t>bFGF</t> productivity of cancer cells transfected with VEGF cDNA. ▪ parental cells; □ transfected clones. The levels of VEGF and bFGF contained in conditioned medium and cell extract, respectively, were determined by sandwich ELISA. Sample values were plotted against a recombinant VEGF or bFGF standard curve (mean ± SE, n = 3). ND: not detectable. QG90, RPMI4788, MCF-7 and their transfected clones were cultured in RPMI1640 containing 10% FCS (normal). Hormones-stripped FCS with (E 2 +) or without (E 2 −) 10 −8 M 17 β-oestradiol was used for MCF-7 and its transfected clone, M2–24-G10.
    Vegf, supplied by PeproTech, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeproTech human recombinant vegf a
    VEGFR2/Stat-3 pathway is involved in <t>VEGF-A-induced</t> resistance to anti-EGFR therapy ( A ) Western blot analysis performed on SW48 colon cancer cell line. Cells were stimulated with human recombinant VEGF-A (5 ng/mL) and with or without axitinib or STA-21 during 24 hours. Cetuximab was added the following day for 24 hours. α-tubulin was used as loading control (Co: Control). ( B ) Cell proliferation analyzed by crystal violet staining. Colon cancer cell lines were incubated or not with human recombinant VEGF-A (5 ng/mL) and STA-21 (10 μM) or axitinib (500 pM) were concomitantly added. Cetuximab (500μg/mL) was added the following day in culture medium and cell death was analyzed 7 days later. (* p
    Human Recombinant Vegf A, supplied by PeproTech, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (a) VEGF and (b) bFGF productivity of cancer cells transfected with VEGF cDNA. ▪ parental cells; □ transfected clones. The levels of VEGF and bFGF contained in conditioned medium and cell extract, respectively, were determined by sandwich ELISA. Sample values were plotted against a recombinant VEGF or bFGF standard curve (mean ± SE, n = 3). ND: not detectable. QG90, RPMI4788, MCF-7 and their transfected clones were cultured in RPMI1640 containing 10% FCS (normal). Hormones-stripped FCS with (E 2 +) or without (E 2 −) 10 −8 M 17 β-oestradiol was used for MCF-7 and its transfected clone, M2–24-G10.

    Journal: International Journal of Experimental Pathology

    Article Title: Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

    doi: 10.1046/j.1365-2613.1999.00122.x

    Figure Lengend Snippet: (a) VEGF and (b) bFGF productivity of cancer cells transfected with VEGF cDNA. ▪ parental cells; □ transfected clones. The levels of VEGF and bFGF contained in conditioned medium and cell extract, respectively, were determined by sandwich ELISA. Sample values were plotted against a recombinant VEGF or bFGF standard curve (mean ± SE, n = 3). ND: not detectable. QG90, RPMI4788, MCF-7 and their transfected clones were cultured in RPMI1640 containing 10% FCS (normal). Hormones-stripped FCS with (E 2 +) or without (E 2 −) 10 −8 M 17 β-oestradiol was used for MCF-7 and its transfected clone, M2–24-G10.

    Article Snippet: The captured VEGF and bFGF were detected by rabbit neutralizing polyclonal antibodies against VEGF (PeproTech, Rocky Hill, NJ) and bFGF (R & D Systems, Minneapolis, MN), respectively, followed by the addition of horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (KPL, Gaithersburg, MD).

    Techniques: Transfection, Clone Assay, Sandwich ELISA, Recombinant, Cell Culture

    Neutralization by anti-VEGF rabbit polyclonal antibodies (pAb) of the HUVE cell growth stimulatory activity secreted by transfected clones. Conditioned medium from each clone was added to HUVE cells at a concentration of 50% in the absence (□) or presence (▪) of anti-VEGF pAb. After the incubation for 4 days, HUVE cell growth was determined by MTT assay. Results are presented as the percentage growth relative to the fresh medium-treated control and are the average of triplicate determinations (mean ± SE). Additional control cells were incubated with 2.5 ng/ml of human recombinant VEGF165 (hr-VEGF165) or 4 ng/ml of human recombinant bFGF (hr-bFGF).

    Journal: International Journal of Experimental Pathology

    Article Title: Vascular endothelial growth factor overproduced by tumour cells acts predominantly as a potent angiogenic factor contributing to malignant progression

    doi: 10.1046/j.1365-2613.1999.00122.x

    Figure Lengend Snippet: Neutralization by anti-VEGF rabbit polyclonal antibodies (pAb) of the HUVE cell growth stimulatory activity secreted by transfected clones. Conditioned medium from each clone was added to HUVE cells at a concentration of 50% in the absence (□) or presence (▪) of anti-VEGF pAb. After the incubation for 4 days, HUVE cell growth was determined by MTT assay. Results are presented as the percentage growth relative to the fresh medium-treated control and are the average of triplicate determinations (mean ± SE). Additional control cells were incubated with 2.5 ng/ml of human recombinant VEGF165 (hr-VEGF165) or 4 ng/ml of human recombinant bFGF (hr-bFGF).

    Article Snippet: The captured VEGF and bFGF were detected by rabbit neutralizing polyclonal antibodies against VEGF (PeproTech, Rocky Hill, NJ) and bFGF (R & D Systems, Minneapolis, MN), respectively, followed by the addition of horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (KPL, Gaithersburg, MD).

    Techniques: Neutralization, Activity Assay, Transfection, Clone Assay, Concentration Assay, Incubation, MTT Assay, Recombinant

    VEGFR2/Stat-3 pathway is involved in VEGF-A-induced resistance to anti-EGFR therapy ( A ) Western blot analysis performed on SW48 colon cancer cell line. Cells were stimulated with human recombinant VEGF-A (5 ng/mL) and with or without axitinib or STA-21 during 24 hours. Cetuximab was added the following day for 24 hours. α-tubulin was used as loading control (Co: Control). ( B ) Cell proliferation analyzed by crystal violet staining. Colon cancer cell lines were incubated or not with human recombinant VEGF-A (5 ng/mL) and STA-21 (10 μM) or axitinib (500 pM) were concomitantly added. Cetuximab (500μg/mL) was added the following day in culture medium and cell death was analyzed 7 days later. (* p

    Journal: Oncotarget

    Article Title: Does bevacizumab impact anti-EGFR therapy efficacy in metastatic colorectal cancer?

    doi: 10.18632/oncotarget.7008

    Figure Lengend Snippet: VEGFR2/Stat-3 pathway is involved in VEGF-A-induced resistance to anti-EGFR therapy ( A ) Western blot analysis performed on SW48 colon cancer cell line. Cells were stimulated with human recombinant VEGF-A (5 ng/mL) and with or without axitinib or STA-21 during 24 hours. Cetuximab was added the following day for 24 hours. α-tubulin was used as loading control (Co: Control). ( B ) Cell proliferation analyzed by crystal violet staining. Colon cancer cell lines were incubated or not with human recombinant VEGF-A (5 ng/mL) and STA-21 (10 μM) or axitinib (500 pM) were concomitantly added. Cetuximab (500μg/mL) was added the following day in culture medium and cell death was analyzed 7 days later. (* p

    Article Snippet: In some cases cells were treated with human recombinant VEGF-A (Peprotech, Neuilly sur Seine, France) at 0,5 or 5 ng/mL the day before cetuximab treatment.

    Techniques: Western Blot, Recombinant, Staining, Incubation

    VEGF-A is increased in patients’ serum during anti-VEGF therapy VEGF-A serum level from mCRC patients ( n = 26) treated with FOLFOX/bevacizumab chemotherapy protocol red lines) compared to patients ( n = 12) treated with chemotherapy alone (blue lines). Assays were performed before and 15 days after bevacizumab injection and analyzed by ELISA. (* p

    Journal: Oncotarget

    Article Title: Does bevacizumab impact anti-EGFR therapy efficacy in metastatic colorectal cancer?

    doi: 10.18632/oncotarget.7008

    Figure Lengend Snippet: VEGF-A is increased in patients’ serum during anti-VEGF therapy VEGF-A serum level from mCRC patients ( n = 26) treated with FOLFOX/bevacizumab chemotherapy protocol red lines) compared to patients ( n = 12) treated with chemotherapy alone (blue lines). Assays were performed before and 15 days after bevacizumab injection and analyzed by ELISA. (* p

    Article Snippet: In some cases cells were treated with human recombinant VEGF-A (Peprotech, Neuilly sur Seine, France) at 0,5 or 5 ng/mL the day before cetuximab treatment.

    Techniques: Injection, Enzyme-linked Immunosorbent Assay

    VEGF-A can inhibit cetuximab cytoxicity in vitro ( A ) Western Blot analysis showing VEGFR1, VEGFR2 and EGFR expression. HCS-70 was used as loading control and as a reference for EGFR quantification (a.u: arbitrary unit). ( B ) Cell proliferation analyzed by crystal violet staining. SW48, Caco-2 and Colo320 colon cancer cell lines were incubated or not with increasing dose of human recombinant VEGF-A (0,5 or 5 ng/mL). Cetuximab (500 μg/mL) was added the following day in culture medium and cell death was analyzed 7 days later. ( C ) Annexin V/7AAD staining. Cells were incubated with VEGF-A 5 ng/mL. Cetuximab 500 μg/mL was added the following day. Cell death was evaluated 24 hours after cetuximab was added, AnnexinV positive cells are in black boxes, double positive cells are in white boxes (* p

    Journal: Oncotarget

    Article Title: Does bevacizumab impact anti-EGFR therapy efficacy in metastatic colorectal cancer?

    doi: 10.18632/oncotarget.7008

    Figure Lengend Snippet: VEGF-A can inhibit cetuximab cytoxicity in vitro ( A ) Western Blot analysis showing VEGFR1, VEGFR2 and EGFR expression. HCS-70 was used as loading control and as a reference for EGFR quantification (a.u: arbitrary unit). ( B ) Cell proliferation analyzed by crystal violet staining. SW48, Caco-2 and Colo320 colon cancer cell lines were incubated or not with increasing dose of human recombinant VEGF-A (0,5 or 5 ng/mL). Cetuximab (500 μg/mL) was added the following day in culture medium and cell death was analyzed 7 days later. ( C ) Annexin V/7AAD staining. Cells were incubated with VEGF-A 5 ng/mL. Cetuximab 500 μg/mL was added the following day. Cell death was evaluated 24 hours after cetuximab was added, AnnexinV positive cells are in black boxes, double positive cells are in white boxes (* p

    Article Snippet: In some cases cells were treated with human recombinant VEGF-A (Peprotech, Neuilly sur Seine, France) at 0,5 or 5 ng/mL the day before cetuximab treatment.

    Techniques: In Vitro, Western Blot, Expressing, Staining, Incubation, Recombinant

    Treatment with EA inhibits T24 bladder cancer cell invasion in response to VEGF-A, but not to EGF. Invasion of T24 cells (2 × 10 5 cells/chamber, 2 h incubation), non-stimulated (CTR) or exposed to EA IC 25 (10 µM) in response to VEGF-A (50 ng/mL) or to EGF (50 ng/mL), was tested in Boyden chambers containing matrigel coated filters. Invading cells were counted in six random microscopic fields for each experimental condition. The histogram represents the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis performed by one-way ANOVA, followed by Bonferroni’s post-test for multiple comparison, were as follows: VEGF-A vs. CTR or EA, p

    Journal: Nutrients

    Article Title: Ellagic Acid Inhibits Bladder Cancer Invasiveness and In Vivo Tumor Growth

    doi: 10.3390/nu8110744

    Figure Lengend Snippet: Treatment with EA inhibits T24 bladder cancer cell invasion in response to VEGF-A, but not to EGF. Invasion of T24 cells (2 × 10 5 cells/chamber, 2 h incubation), non-stimulated (CTR) or exposed to EA IC 25 (10 µM) in response to VEGF-A (50 ng/mL) or to EGF (50 ng/mL), was tested in Boyden chambers containing matrigel coated filters. Invading cells were counted in six random microscopic fields for each experimental condition. The histogram represents the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis performed by one-way ANOVA, followed by Bonferroni’s post-test for multiple comparison, were as follows: VEGF-A vs. CTR or EA, p

    Article Snippet: Invasion medium (200 μL), containing or not human VEGF-A (50 ng/mL; Peprotech, Rocky Hill, NJ, USA) or, in selected experiments, epidermal growth factor (EGF) (50 ng/mL; Peprotech), was added to the lower compartment of the chambers.

    Techniques: Incubation

    Treatment with EA inhibits migration of UM-UC-3 cells in response to VEGF-A but not to EGF. Migration of UM-UC-3 cells (2 × 10 5 cells/chamber, 18 h incubation), non-stimulated (CTR) or exposed to EA IC 25 (20 µM) in response to VEGF-A or EGF (50 ng/mL) was tested in Boyden chambers containing gelatin coated filters. Migrating cells were counted in six random microscopic fields for each experimental condition. The histogram represents the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test, were as follows: VEGF-A vs. CTR or EA, p

    Journal: Nutrients

    Article Title: Ellagic Acid Inhibits Bladder Cancer Invasiveness and In Vivo Tumor Growth

    doi: 10.3390/nu8110744

    Figure Lengend Snippet: Treatment with EA inhibits migration of UM-UC-3 cells in response to VEGF-A but not to EGF. Migration of UM-UC-3 cells (2 × 10 5 cells/chamber, 18 h incubation), non-stimulated (CTR) or exposed to EA IC 25 (20 µM) in response to VEGF-A or EGF (50 ng/mL) was tested in Boyden chambers containing gelatin coated filters. Migrating cells were counted in six random microscopic fields for each experimental condition. The histogram represents the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test, were as follows: VEGF-A vs. CTR or EA, p

    Article Snippet: Invasion medium (200 μL), containing or not human VEGF-A (50 ng/mL; Peprotech, Rocky Hill, NJ, USA) or, in selected experiments, epidermal growth factor (EGF) (50 ng/mL; Peprotech), was added to the lower compartment of the chambers.

    Techniques: Migration, Incubation

    Inhibitory effect of EA on UM-UC-3 cell invasion in response to VEGF-A. Matrigel invasion assay. Invasion of UM-UC-3 cells (2 × 10 5 cells/chamber, 4 h incubation), non-stimulated (CTR) or exposed to EA IC 25 (20 µM) in response to VEGF-A (50 ng/mL) was tested in Boyden chambers containing matrigel coated filters ( A , B ) or by spheroid invasion assay ( C , D ). For matrigel invasion test, invading cells were counted in six random microscopic fields for each experimental condition. Histogram represents the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test, were as follows: VEGF-A vs. CTR or EA, p

    Journal: Nutrients

    Article Title: Ellagic Acid Inhibits Bladder Cancer Invasiveness and In Vivo Tumor Growth

    doi: 10.3390/nu8110744

    Figure Lengend Snippet: Inhibitory effect of EA on UM-UC-3 cell invasion in response to VEGF-A. Matrigel invasion assay. Invasion of UM-UC-3 cells (2 × 10 5 cells/chamber, 4 h incubation), non-stimulated (CTR) or exposed to EA IC 25 (20 µM) in response to VEGF-A (50 ng/mL) was tested in Boyden chambers containing matrigel coated filters ( A , B ) or by spheroid invasion assay ( C , D ). For matrigel invasion test, invading cells were counted in six random microscopic fields for each experimental condition. Histogram represents the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test, were as follows: VEGF-A vs. CTR or EA, p

    Article Snippet: Invasion medium (200 μL), containing or not human VEGF-A (50 ng/mL; Peprotech, Rocky Hill, NJ, USA) or, in selected experiments, epidermal growth factor (EGF) (50 ng/mL; Peprotech), was added to the lower compartment of the chambers.

    Techniques: Invasion Assay, Incubation

    Treatment with EA reduces VEGFR-2 expression. ( A ) VEGF-A levels released in tumor cell culture supernatants. Quantification of the amount of VEGF-A in the concentrated supernatants of bladder cancer cell lines was performed using Maxisorp Nunc immunoplates coated with goat anti-VEGF-A IgGs. Results are the mean (± SD) of three independent determinations; ( B ) Immunoblot analysis of VEGFR-2. Western blot analysis of the levels of VEGFR-2 expressed in control (CTR) and in bladder cancer cell lines, treated with a vehicle (CTR) or exposed to EA for 24 h, at concentrations in the range of IC 50 values for each cell line (i.e., 20 µM, T24; 40 µM, UM-UC-3; 27 µM 5637; 60 µM HT-1376). HUVEC were loaded as a positive control and β-actin as a loading control; ( C ) Densitometric analysis. The relative levels of VEGFR-2 were calculated by densitometric analysis and normalized using β-actin expression in each sample. The histogram represents the ratios between the optical densities (O.D.) of VEGFR-2 in CTR or EA treated groups and β-actin. Results are the mean (±SD) of three independent experiments. Student’s t -test analysis: EA vs. CTR, p

    Journal: Nutrients

    Article Title: Ellagic Acid Inhibits Bladder Cancer Invasiveness and In Vivo Tumor Growth

    doi: 10.3390/nu8110744

    Figure Lengend Snippet: Treatment with EA reduces VEGFR-2 expression. ( A ) VEGF-A levels released in tumor cell culture supernatants. Quantification of the amount of VEGF-A in the concentrated supernatants of bladder cancer cell lines was performed using Maxisorp Nunc immunoplates coated with goat anti-VEGF-A IgGs. Results are the mean (± SD) of three independent determinations; ( B ) Immunoblot analysis of VEGFR-2. Western blot analysis of the levels of VEGFR-2 expressed in control (CTR) and in bladder cancer cell lines, treated with a vehicle (CTR) or exposed to EA for 24 h, at concentrations in the range of IC 50 values for each cell line (i.e., 20 µM, T24; 40 µM, UM-UC-3; 27 µM 5637; 60 µM HT-1376). HUVEC were loaded as a positive control and β-actin as a loading control; ( C ) Densitometric analysis. The relative levels of VEGFR-2 were calculated by densitometric analysis and normalized using β-actin expression in each sample. The histogram represents the ratios between the optical densities (O.D.) of VEGFR-2 in CTR or EA treated groups and β-actin. Results are the mean (±SD) of three independent experiments. Student’s t -test analysis: EA vs. CTR, p

    Article Snippet: Invasion medium (200 μL), containing or not human VEGF-A (50 ng/mL; Peprotech, Rocky Hill, NJ, USA) or, in selected experiments, epidermal growth factor (EGF) (50 ng/mL; Peprotech), was added to the lower compartment of the chambers.

    Techniques: Expressing, Cell Culture, Western Blot, Positive Control

    Inhibitory effect of EA on 5637 and HT-1376 cell invasion in response to VEGF-A. Invasion of 5637 ( A , C ) and HT-1376 ( B , D ) cells (2 × 10 5 cells/chamber, 18 h incubation), non-stimulated (CTR) or exposed to EA IC 25 of each cell line (13.5 µM for 5637 cells and 30 µM for HT-1376) in response to VEGF-A (50 ng/mL) was tested in Boyden chambers containing matrigel coated filters. Invading cells were counted in six random microscopic fields for each experimental condition. Histograms represent the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test, were as follows for both cell lines: VEGF-A vs. CTR or EA, p

    Journal: Nutrients

    Article Title: Ellagic Acid Inhibits Bladder Cancer Invasiveness and In Vivo Tumor Growth

    doi: 10.3390/nu8110744

    Figure Lengend Snippet: Inhibitory effect of EA on 5637 and HT-1376 cell invasion in response to VEGF-A. Invasion of 5637 ( A , C ) and HT-1376 ( B , D ) cells (2 × 10 5 cells/chamber, 18 h incubation), non-stimulated (CTR) or exposed to EA IC 25 of each cell line (13.5 µM for 5637 cells and 30 µM for HT-1376) in response to VEGF-A (50 ng/mL) was tested in Boyden chambers containing matrigel coated filters. Invading cells were counted in six random microscopic fields for each experimental condition. Histograms represent the arithmetic mean values of migrated cells/microscopic field ± SD of three independent determinations. Results of the statistical analysis using one-way ANOVA, followed by Bonferroni’s post-test, were as follows for both cell lines: VEGF-A vs. CTR or EA, p

    Article Snippet: Invasion medium (200 μL), containing or not human VEGF-A (50 ng/mL; Peprotech, Rocky Hill, NJ, USA) or, in selected experiments, epidermal growth factor (EGF) (50 ng/mL; Peprotech), was added to the lower compartment of the chambers.

    Techniques: Incubation

    Study design. E-MNCs are generated in a hematopoietic stem cell expansion medium containing recombinant human SCF, recombinant human Flt-3 ligand, recombinant human TPO, recombinant human VEGF, and recombinant human IL-6. CT = computed tomography, MRI = magnetic resonance imaging, PBMNCs = peripheral blood mononuclear cells, E-MNC = effective mononuclear cell, CPC = cell-processing center.

    Journal: Medicine

    Article Title: Phase 1 clinical study of cell therapy with effective-mononuclear cells (E-MNC) for radiogenic xerostomia (first-in-human study) (FIH study on E-MNC therapy for radiogenic xerostomia)

    doi: 10.1097/MD.0000000000020788

    Figure Lengend Snippet: Study design. E-MNCs are generated in a hematopoietic stem cell expansion medium containing recombinant human SCF, recombinant human Flt-3 ligand, recombinant human TPO, recombinant human VEGF, and recombinant human IL-6. CT = computed tomography, MRI = magnetic resonance imaging, PBMNCs = peripheral blood mononuclear cells, E-MNC = effective mononuclear cell, CPC = cell-processing center.

    Article Snippet: [ ] Briefly, PBMNCs are cultured in serum-free medium (Stemline II Hematopoietic Stem Cell Expansion Medium; Sigma Aldrich) with recombinant human stem cell factor (SCF), recombinant human Fms-related tyrosine kinase 3 (Flt-3) ligand, recombinant human thrombopoietin (TPO), recombinant human vascular endothelial growth factor (VEGF), and recombinant human interleukin-6 (IL-6) (all from Peprotech, Rocky Hill, NJ), which is modified human quality- and quantify-controlled culture system as described in our previous work.

    Techniques: Generated, Recombinant, Computed Tomography, Magnetic Resonance Imaging