Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) The extracellular domain of mouse VEGFR-3 was immobilized on microtiter wells and incubated with the X6 phage display library. Bar graph shows enrichment in the number of phage recovered [in transducing units (TU)] after consecutive rounds of selection (I, II, and III). (*) Round I was not quantified to prevent the loss of phage displaying unique peptides. ( B ) Peptide identified by sequencing phage bound to VEGFR-3 (round III) ( n , number of phages sequenced). ( C and D ) Binding of control phage Fd (white bars) and phage PCAIWF (B, black bars) and WVCSGG (C, black bars) to VEGF receptors and co-receptors immobilized on microtiter wells. ( E and F ) Inhibition of phage PCAIWF (E) or WVCSGG (F) binding to immobilized VEGFR-3 by synthetic peptide PCAIWF or control peptide (CARAC). The minus sign indicates that no synthetic peptide was added to the assay. ( G ) Dose-response assay. Phage PCAIWF was incubated with immobilized VEGFR-3 in the presence of synthetic peptides PCAIWF, PSAIWF, or CARAC (control). Percentage relative to phage binding in the absence of competing peptide. In all cases, bars represent means ± SEM from triplicate plating. Statistics, Student’s t test (** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Incubation, Selection, Sequencing, Binding Assay, Control, Inhibition

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Binding of phage PCAIWF to immobilized VEGFR-3 in the presence or absence of VEGF-A or VEGF-C (10 ng/ml). ( B ) Binding of phage PCAIWF to immobilized VEGFR-3 in the presence of increasing concentrations of VEGF-C. Percentage relative to phage binding in the absence of VEGF-C. ( C ) Cartoon showing the three-dimensional structure of the complex VEGF-C (red) bound to VEGFR-2 IgD2-3 (shown in orange and green, respectively) (Protein Data Bank #2X1W). ( D ) Analysis by SDS–polyacrylamide gel electrophoresis of purified recombinant IgD2 and IgD2-3 proteins containing the ligand-binding domain of VEGFR-3. ( E ) Binding of phage PCAIWF to VEGFR-3 and its recombinant Ig domains immobilized on microtiter wells in the presence or absence of the synthetic peptide PCAIWF or its scramble version, IFCAPW (100 μg/ml). Phage binding was quantified by FLISA using an anti-bacteriophage sera. ( F ) Binding of VEGF-C to microtiter wells coated with immobilized recombinant ligand binding domains IgD2 and IgD2-3 of VEGFR-3 in the presence or absence of synthetic peptides PCAIWF and WVCSGG or the scramble control peptide (IFCAPW). For phage experiments (A and B), bars represent mean ± SEM from triplicate plating; for FLISA assays ( E to G ), bars represent means ± SEM from duplicate wells. Statistics, Student’s t test [not significant (N.S.), P > 0.05; * P ≤ 0.05 and *** P ≤ 0.001].

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Binding Assay, Polyacrylamide Gel Electrophoresis, Purification, Recombinant, Ligand Binding Assay, Fluorophore-linked Immunoabsorbent Assay, Control

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Representation of the VEGF family, their receptors, and pattern of interaction. ( B to F ) Recombinant proteins for the human VEGFR-3 (B), VEGFR-2 (C and E), and VEGFR-1 (D and F) extracellular domains were immobilized on microtiter wells and incubated with the human ligands VEGF-C (B and C), PlGF (D), and VEGF-A (E and F) in the presence or absence of synthetic peptides PCAIWF and PSAIWF or the scramble control peptide (IFCAPW). Growth factors bound to the wells were quantified by FLISA using immunospecific antibodies and fluorescent detection. Bars represent means ± SEM from duplicate wells. Statistics, analysis of variance (ANOVA) (Tukey’s multiple comparison test) (* P ≤ 0.05; ** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Recombinant, Incubation, Control, Fluorophore-linked Immunoabsorbent Assay, Comparison

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Immunoblot analysis of phosphorylated and total forms of ERK1/2 in LECs incubated with VEGF-A, VEGF-C, or FGF (100 ng/ml) in the presence or absence of peptide PCAIWF or scramble (IFCAPW) (30 μg/ml). ( B ) Ratio of fluorescent intensity for phosphorylated and total ERK1/2. Bars represent means ± SEM from three independent measurements of the immunoblot membrane. Two independent experiments were performed with similar results. Bars represent means ± SEM from triplicate readings. Statistics, ANOVA (Tukey’s multiple comparison test) (* P ≤ 0.05).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Western Blot, Incubation, Membrane, Comparison

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Tube formation by HUVECs in Matrigel induced by VEGF or VEGF-C in the presence or absence of peptide PCAIWF or scramble (500 μg/ml, embedded in the Matrigel layer). ( B ) Number of tubes formed between endothelial cells. Bars represent means ± SEM from triplicate wells. Statistics, Student’s t test (* P ≤ 0.05). Two independent experiments were performed with similar results.

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques:

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: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: Autophagy is induced in MT cells. ( A ) Mutant UQCRB-expressing cell lines were examined by Western blotting using anti-UQCRB and anti-Myc antibodies. ( B ) HEK293 and MT cells treated with either 10% serum-containing medium or serum-free medium for 24 h and then analyzed by Western blot analysis using antibodies against LC3B and p62. shows treatment of either 10% serum-containing medium or serum-free medium for 4 h. ( C ) Confocal microscopy images of HEK293 and MT cells treated with serum-free medium for 24 h after immunostaining with anti-LC3 antibody. Scale bar, 50 μm. ( D ) Western blot analysis results of LC3B and p62 levels in cells after treatment with 5 μM chloroquine (CQ) treatment for 0–6 h. ( E ) HEK293 and MT cells were treated with either deionized water (DIW) or CQ for 6 h. Cell extracts were subjected to Western blotting using LC3B and p62 antibodies. The densitometric value of LC3-II was quantitated based on Western blot data and normalized to LC3-I levels. ( F ) Confocal microscopy images of HEK293 and MT cells transfected with mRFP-GFP-LC3 for 24 h. Scale bar, 20 μm. ( G ) Numbers of mRFP-LC3 puncta were counted in HEK293 and MT cells ( n = 8). The relative band intensities value was normalized to β-actin (loading control). The normalized protein levels are shown under each band. Data are presented as the mean ± SD; statistical significance was assessed using an unpaired t -test. ns, p > 0.05.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Mutagenesis, Expressing, Western Blot, Confocal Microscopy, Immunostaining, Transfection, Control

Journal: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: TRPML1-induced lysosomal Ca 2+ release and TFEB activation are increased in MT cells. HEK293 and MT cells were transfected with GCaMP3-TRPML1 encoding a lysosome-specific Ca 2+ probe, 40 µM ML-SA1, for 120 s. ( A ) Confocal microscopy images of GCaMP green fluorescence showing HEK293 and ( B ) MT cells. ML-SA1 treatment for 120 s. ( C ) Confocal microscopy images of HEK293 and MT cells transfected with EGFP-TFEB and treated with the DMSO control or A1938 for 6 h. ( D ) The number of cells with a nuclear (Nuc) or cytoplasmic (Cyt) TFEB localization ratio is represented in the graph. Scale bar, 20 μm. Statistical significance was assessed using Student’s t -test. ** p < 0.01.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Activation Assay, Transfection, Confocal Microscopy, Fluorescence, Control

Journal: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: Lysosome activity in MT cells with or without A1938 treatment. ( A ) Confocal microscopy images and ( B ) numbers of acidic lysosome red puncta were counted in HEK293 and MT cells ( n = 15). The cells were treated with DMSO as a control or 30 µM A1938 for 24 h and then stained with LTR. Scale bar, 20 μm. ( C ) Confocal microscopy images of MT cells treated with DMSO as a control or 30 µM A1938 for 24 h, then the live cells were stained with 2 μg/mL acridine orange for 25 min and fixed. Scale bar, 20 μm. ( D ) Images of HEK293 and MT cell extracts treated with the DMSO control or 30 µM A1938 for 24 h in a serum-free medium, then subjected to Western blot analysis using antibodies against LAMP1. ( E ) Intensities of the LAMP1 immunoblot bands were normalized to β-actin expression. Western blot analysis out of three independent experiments. Statistical significance was assessed using Student’s t -test. ns, p > 0.05, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Activity Assay, Confocal Microscopy, Control, Staining, Western Blot, Expressing

Journal: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: A1938 regulates autophagy flux by suppression of mROS generation in MT cells. ( A ) Cells treated with A1938 for 24 h in a serum-free medium. The relative band intensities value was normalized to β-actin (loading control). The normalized protein levels are shown under each band. ( B ) Confocal microscopy images of HEK293 and MT cells transfected with mRFP-GFP-LC3 and treated with DMSO, A1938, or Mito-Tempo (mTP) for 24 h. Scale bar, 20 μm. Quantification of data indicating red puncta (autolysosome) versus yellow puncta (autophagosome) is shown in ( C ). Values are the means ± SEM; statistical significance was assessed using a paired t -test. n = 10 cells, ns, p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Control, Confocal Microscopy, Transfection