Journal: Cell Communication and Signaling : CCS

Article Title: Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

doi: 10.1186/s12964-024-01606-w

Figure Lengend Snippet: Essential role of EMCN in facilitating VEGFR2 and AP2 interaction and clathrin recruitment. A Colocalization of clathrin HC (heavy chain) (green) and VEGFR2 (red) were visualized in control HRECs with or without VEGF165 (10 ng/ml). Examples of colocalization of VEGFR2 and clathrin HC (white arrowhead), and clathrin (white arrow) were shown in magnified view. Bar = 10 µm B Fraction of VEGFR2 that colocalized with clathrin was quantified by Image J CoJAP plugin. Student t-test was used for the comparison. * P < 0.05, n = 3. C Colocalization of clathrin HC (heavy chain) (green) and VEGFR2 (red) were visualized in siNT or siEMCN HRECs with VEGF165 (10 ng/ml) stimulation. Examples of colocalization of VEGFR2 and clathrin HC (white arrowheads), and VEGFR2 (white arrow) were shown in magnified view. Bar = 10 µm D Fraction of VEGFR2 that colocalized with clathrin in siNT and siEMCN HRECs was quantified. Student t-test was used for the comparison. ** P < 0.01, n = 3. E Colocalization of clathrin HC (heavy chain) (green) and AP2 (red) were visualized in control in siNT and siEMCN HRECs with VEGF(10 ng/ml) stimulation. Examples of colocalization of clathrin HC and AP2 (white arrowhead) were shown in magnified view. Bar = 10 µm ( F ) Fraction of clathrin that colocalized with AP2 in siNT and siEMCN HRECs with VEGF stimulation were quantified. Student t-test was used for the comparison. P > 0.05, n = 3. G EMCN is required for interaction between VEGFR2 and AP2A2. HRECs were lysed and VEGFR2 that co-immunoprecipitated with AP2A2 in the presence and absence of EMCN was observed. n = 3

Article Snippet: Recombinant human VEGF165 (#293-VE), PlGF-2 (#6837-PL), and FGF2 (#233-FB) were purchased from R&D Systems.

Techniques: Control, Comparison, Immunoprecipitation

Journal: Cell Communication and Signaling : CCS

Article Title: Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

doi: 10.1186/s12964-024-01606-w

Figure Lengend Snippet: EMCN does not modulate VEGF165 or PIGF-induced endothelial migration or VEGFR1 internalization. A EMCN knockdown did not affect PlGF-2-induced HREC migration. HRECs were transfected with either siNT or siEMCN, mechanically scratched, stimulated with PlGF-2 (10 ng/ml) or VEGF165 (10 ng/ml), and the resulting cell migration was quantified by image analysis (left). ** P < 0.01, n = 6 or 9. Representative images of each group at time zero (white dashed line) and 15 h time (yellow dashed line) points (right). The scale bar represents 500 µm. B Illustration of the cell surface receptor internalization assay. Growth factors bind to its cell surface receptors and induce receptor internalization. Cell surface proteins are biotinylated, the cell surface fraction is separated using avidin resin, and western blot analysis were used to analyze the fraction of receptors remaining at the cell surface. C HRECs incubated in serum-free media were stimulated with VEGF165 (10 ng/ml) for 30 min with and without EMCN knockdown, and cell surface membrane-bound VEGFR1 (mVEGFR1) levels were analyzed by western blot analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, n = 6. One-way ANOVA was used for statistical analysis

Article Snippet: Recombinant human VEGF165 (#293-VE), PlGF-2 (#6837-PL), and FGF2 (#233-FB) were purchased from R&D Systems.

Techniques: Migration, Knockdown, Transfection, Cell Surface Receptor Assay, Avidin-Biotin Assay, Western Blot, Incubation, Membrane

Journal: Cell Communication and Signaling : CCS

Article Title: Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

doi: 10.1186/s12964-024-01606-w

Figure Lengend Snippet: EMCN is not required for FGF2-induced HREC cell migration or FGFR1 internalization. A EMCN knockdown did not affect FGF2-induced HREC migration. HRECs were transfected with either siNT or siEMCN, incubated in serum-free media for 8 h, mechanically scratched, and stimulated with FGF2 (10 ng/ml) or VEGF165 (10 ng/ml). Quantification of cell migration for all the treatment groups based on image analysis (left). Student t-test was used for comparisons within groups. * P < 0.05, n = 8. Representative images of each group at time zero (white dashed line) and 15 h time (yellow dashed line) points (right). The scale bar represents 500 µm. B EMCN knockdown did not affect FGF2-induced FGFR1 internalization in HREC. Serum-starved HRECs were stimulated with FGF2 for 45 min, and then the cell surface proteins were isolated and visualized by western blot. Quantification of FGFR1 at the cell surface from all treatment groups by western blot analysis (left). Student-t test was used for statistical analysis. * P < 0.05, n = 7. A representative western blot for all treatment groups (right). C EMCN does not interact with VEGFR1 or FGFR1 in HRECs. HRECs overexpressing myc-tagged EMCN were lysed, and cell surface receptors that co-immunoprecipitated with EMCN were observed. n = 3. Note that the IgG and Myc groups were overexposed together for the better detection of the different receptors because of the low protein levels, while the input groups were kept at a lower exposure

Article Snippet: Recombinant human VEGF165 (#293-VE), PlGF-2 (#6837-PL), and FGF2 (#233-FB) were purchased from R&D Systems.

Techniques: Migration, Knockdown, Transfection, Incubation, Isolation, Western Blot, Immunoprecipitation

Journal: Cell Communication and Signaling : CCS

Article Title: Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2

doi: 10.1186/s12964-024-01606-w

Figure Lengend Snippet: EMCN knockdown inhibits VEGF121 induced VEGFR2 internalization and HRECs migration similar to VEGF165. A Both VEGF165 (10 ng/ml)- and VEGF121 (7.29 ng/ml)-induced migration were inhibited with EMCN knockdown. Quantification of cell migration by image analysis is shown (left). Student-t test was used for comparisons between groups. * P < 0.05, ** P < 0.01, n = 10. Representative images of each group at time zero (white dashed line) and 15 h time (yellow dashed line) points. The scale bar represents 500 µm. B Schematic representation of VEGFA isoforms, VEGF165 and VEGF121. C HRECs were treated with siEMCN stimulated with VEGF121 (7.29 ng/ml) for a time course of up to 120 min. VEGF121 induced significant VEGFR2 endocytosis after 60 min of stimulation, except when EMCN was knockdown. One-way ANOVA was used for comparation within group. Student-t test was used for comparation between siNT and siEMCN at the same time point. # P < 0.05, * P < 0.05, *** P < 0.001, **** P < 0.0001, n = 3. D Representative image of the western blot for VEGFR2 internalization in both siNT and siEMCN groups. CD31 was blotted as cell surface fraction loading control

Article Snippet: Recombinant human VEGF165 (#293-VE), PlGF-2 (#6837-PL), and FGF2 (#233-FB) were purchased from R&D Systems.

Techniques: Knockdown, Migration, Western Blot, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: 17Beta-estradiol up-regulates vascular endothelial growth factor receptor-2 expression in human myometrial microvascular endothelial cells: role of estrogen receptor-alpha and -beta.

doi: 10.1210/jc.2001-010588

Figure Lengend Snippet: FIG. 1. Effect of 17-estradiol and progesterone on rhVEGF165 bind- ing to human myometrial MEC. A, Flow cytometry traces showing that (left panel) proliferating MEC bind more rhVEGF165 than qui- escent MEC (basal binding) and (right panel) 17-estradiol (E2), but not progesterone (P4) increased basal rhVEGF165 binding. The neg- ative control represents nonspecific binding. B, Quiescent MEC in- cubated with vehicle, 10 nmol/liter E2, 100 nmol/liter progesterone (P), and 17-estradiol progesterone for 18 h at 37 C in the presence 10 g/liter VEGF were harvested and rhVEGF165 binding measured. Results are mean SEM from four experiments on separate MEC isolates. *, P 0.01.

Article Snippet: Recombinant human (rh) VEGF165, biotinylated rhVEGF165, biotinylated soybean trypsin inhibitor, RDF buffer and avidin-fluorescein isothiocyanate (avidin-FITC) were purchased as a kit from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Binding Assay, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: 17Beta-estradiol up-regulates vascular endothelial growth factor receptor-2 expression in human myometrial microvascular endothelial cells: role of estrogen receptor-alpha and -beta.

doi: 10.1210/jc.2001-010588

Figure Lengend Snippet: FIG. 2. 17-estradiol increased rhVEGF165 binding to myometrial MEC in a time- and dose-dependent manner. A, Time course of rh- VEGF165 binding to MEC incubated with 10 nmol/liter 17-estradiol. B, Concentration-response curve of MEC incubated with or without 17-estradiol for 18 h at 37 C. MFI of rhVEGF165 binding is reported as percentage of control (vehicle-treated cells). Results are mean

Article Snippet: Recombinant human (rh) VEGF165, biotinylated rhVEGF165, biotinylated soybean trypsin inhibitor, RDF buffer and avidin-fluorescein isothiocyanate (avidin-FITC) were purchased as a kit from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Concentration Assay, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: 17Beta-estradiol up-regulates vascular endothelial growth factor receptor-2 expression in human myometrial microvascular endothelial cells: role of estrogen receptor-alpha and -beta.

doi: 10.1210/jc.2001-010588

Figure Lengend Snippet: FIG. 5. 17-estradiol increases VEGFR-2 expression in ER ex- pressing but not in ER myometrial MEC. Quiescent MEC in hu- man serum-free medium were incubated for 18 h at 37 C in the presence of 0 (vehicle), 1 and 10 nmol/liter 17-estradiol and rhVEGF165 binding (f) and VEGFR-2 expression () then measured by flow cytometry. A, Representative flow cytometry histograms of VEGFR-2-FITC fluorescence for ER and ER samples. Negative control is mouse IgG1. B, ER expressing MEC. C, ER MEC. Results are expressed as percentage of control (vehicle only) rhVEGF165 binding or MFI of VEGFR-2 expression. Means SEM from n 4 (B) and n 6 (C) separate MEC isolates. *, P 0.05; **, P 0.01.

Article Snippet: Recombinant human (rh) VEGF165, biotinylated rhVEGF165, biotinylated soybean trypsin inhibitor, RDF buffer and avidin-fluorescein isothiocyanate (avidin-FITC) were purchased as a kit from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Incubation, Binding Assay, Flow Cytometry, Fluorescence, Negative Control, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: 17Beta-estradiol up-regulates vascular endothelial growth factor receptor-2 expression in human myometrial microvascular endothelial cells: role of estrogen receptor-alpha and -beta.

doi: 10.1210/jc.2001-010588

Figure Lengend Snippet: FIG. 6. Antiestrogen ICI 182,780 inhibits 17-estradiol up-regula- tion of rhVEGF165 binding to myometrial MEC and 17-estradiol enhancement of VEGF-stimulated MEC proliferation. A, Quiescent MEC in serum-free medium containing 10 g/liter VEGF were incu- bated for 18 h at 37 C with either 0.1 or 10 nmol/liter 17-estradiol in the presence or absence of 0.01 or 1 mol/liter ICI 182,780 respec- tively and rhVEGF165 binding was then measured. Results are mean SEM from three experiments on separate MEC isolates. *, P 0.01; **, P 0.05. B, Quiescent myometrial MEC (4 103) seeded in triplicate were incubated with 1 mol/liter ICI 182,780 in the pres- ence or absence of 10 nmol/liter 17-estradiol for 6 d at 37 C in phenol-red free M199 medium containing 2 g/liter VEGF and 5% ch-HS and 15% ch-FCS, then MTS reagent added and absorbance measured. Results are presented as the change in absorbance over 6 d expressed as percentage of control MEC (vehicle only). Means ( SEM) from five experiments on separate MEC isolates are shown. *, P 0.01; **, P 0.05.

Article Snippet: Recombinant human (rh) VEGF165, biotinylated rhVEGF165, biotinylated soybean trypsin inhibitor, RDF buffer and avidin-fluorescein isothiocyanate (avidin-FITC) were purchased as a kit from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Control