Journal: Nature Communications

Article Title: VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation

doi: 10.1038/ncomms2683

Figure Lengend Snippet: HEK 293 T cells, PAE/VEGFR2 or HUVECs treated as indicated with VEGF (20 ng ml −1 , 15 min) or Ang1 (200 ng ml −1 , 30 min) or VEGF+Ang1 (Ang 1, 200 ng ml −1 , 15 min, followed by addition of VEGF 20 ng ml −1 , 15 min). Analyses were performed by immunoblotting (blot) on total cell lysates or on immunoprecipitations (IPs) as indicated. All experiments were repeated at least twice. ( a ) HEK 293T cells expressing FLAG-tagged Tie2 (Tie2-FLAG) and WT or D/A mutant V5-tagged VE-PTP (VE-PTP-V5), treated with Ang1. ( b ) PAE/VEGFR2 cells expressing WT or D/A mutant VE-PTP-V5, treated with VEGF. ( c ) PAE/VEGFR2 cells expressing VE-PTP-V5 and Tie2-FLAG, treated with VEGF and Ang1. ( d ) PAE/VEGFR2 cells expressing WT VE-PTP-V5, D/A VE-PTP-V5 and Tie2-FLAG, treated with VEGF and Ang1. ( e ) HUVECs transfected with control or VE-PTP siRNAs, treated with VEGF and Ang1. Quantification of pVEGFR/VEGFR2 levels (right). ( f ) HUVECs transfected with control or Tie2 siRNAs, treated with VEGF. ( g ) HUVECs transfected with control siRNA or with two different Tie1 siRNAs (#1 and #2), and treated with VEGF. Samples were run on the same gel/blot. ( h ) PAE/VEGFR3 cells expressing VE-PTP-V5 and Tie2-FLAG, treated with 200 ng ml −1 VEGFC, for 15 min. ( i ) PAE/VEGFR2 cells expressing VE-PTP-V5 and Tie2-FLAG (Tie2-F), treated with VEGF and Ang1 individually and in combination. Sequential IP was performed first with anti-FLAG antibodies followed by low pH elution and re-IP with anti-V5 antibodies (resulting in VE-PTP enriched samples) as outlined to the upper right. The supernatants (VE-PTP-depleted samples) were used for re-IP with anti-VEGFR2 antibodies. Immunoblotting was performed of total cell lysates (Sample 1; left panel), VE-PTP-V5-enriched samples (Sample 2; middle panel) and VE-PTP-depleted samples (Sample 3; right panel). For the VE-PTP enriched and depleted samples, lanes 1–5 show the results of IP with specific antibodies; control IgG was used for IP of sample 6.

Article Snippet: Antibodies for immunofluorescence: anti-mouse CD31 (BD Biosciences), anti-mouse NG2 (Millipore), anti-mouse Podocalyxin (R&D Systems), anti-mouse Moesin (Abcam), anti-mouse VE-cadherin (BD Biosciences), anti-VE-PTP (hPTPb1-8) (ref. ), anti-mouse VEGFR2 (R&D Systems), anti-human VEGFR2 (R&D Systems), anti-human pY1175 VEGFR2 (Cell Signaling), anti-human Tie2 (R&D Systems), anti-VE-cadherin (BD Biosciences) and anti-pY658 VE-cadherin .

Techniques: Western Blot, Expressing, Mutagenesis, Transfection, Control

Journal: Nature Communications

Article Title: VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation

doi: 10.1038/ncomms2683

Figure Lengend Snippet: ( a ) HUVECs were pretreated with 200 ng ml −1 of Ang1 (30 min), followed by addition of VEGF (20 ng ml −1 , 5 min), alternatively the two ligands were used individually (Ang1 for 30 min and VEGF for 5 min). Cells were immunostained with antibodies against VEGFR2 and Tie2. Scale bars, 20 μm. High-magnification insets show VEGFR2 and Tie2 in merged images; scale bars, 5 μm. ( b ) Quantification of the junctional/cytoplasmic ratio of VEGFR2 (upper) or Tie2 (lower) intensities in a ; mean±s.d., n = 40–50 cells for each condition. ** P <0.01. *** P <0.001, t -test. ( c ) Sparse and confluent HUVECs were treated with 200 ng ml −1 of Ang1 (30 min), 20 ng ml −1 of VEGF (15 min), individually or in combination, followed by immunoprecipitation (IP) with anti-VEGFR2 and blotting for pVEGFR2 and VEGFR2. Quantification of pVEGFR2/VEGFR2 is representative of three independent experiments.

Article Snippet: Antibodies for immunofluorescence: anti-mouse CD31 (BD Biosciences), anti-mouse NG2 (Millipore), anti-mouse Podocalyxin (R&D Systems), anti-mouse Moesin (Abcam), anti-mouse VE-cadherin (BD Biosciences), anti-VE-PTP (hPTPb1-8) (ref. ), anti-mouse VEGFR2 (R&D Systems), anti-human VEGFR2 (R&D Systems), anti-human pY1175 VEGFR2 (Cell Signaling), anti-human Tie2 (R&D Systems), anti-VE-cadherin (BD Biosciences) and anti-pY658 VE-cadherin .

Techniques: Immunoprecipitation

Journal: Nature Communications

Article Title: VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation

doi: 10.1038/ncomms2683

Figure Lengend Snippet: ( a ) HUVECs transfected with control or ve-ptp siRNA were stimulated with VEGF (20 ng ml −1 : 60 min), and with Ang1 (200 ng ml −1 ; 60 min) individually or in combination, followed by PLA for pVEGFR2/VEGFR2 complexes and immunostaining with anti-ZO1 antibodies (green) to visualize cell–cell junctions. Red dots represent PLA products. Scale bars, 10 μm. High-magnification insets show PLA products at junctions; scale bars; 5 μm. ( b ) Quantification of number of pVEGFR2/VEGFR2 PLA products per cell. Mean±s.d., n =45 cells per condition repeated twice. *** P <0.001, t -test. ( c ) Quantification of number of pVEGFR2/VEGFR2 PLA products at junctions per cell. Mean±s.d., n = 45 cells per condition repeated twice. * P <0.05, *** P <0.001, t -test. ( d ) HUVECs were treated with 200 ng ml −1 of Ang1 (30 min), followed by addition of 20 ng ml −1 of VEGF (5 min), alternatively the two ligands were used individually (Ang1 for 30 min and VEGF for 5 min), processed and subjected to PLA using antibodies against VEGFR2 and VE-PTP and immunostaining for ZO1 (green). Scale bars, 20 μm. High-magnification insets show VEGFR2/VE-PTP PLA spots at cell–cell junctions; scale bars, 10 μm. ( e ) Quantification of VEGFR2/VE-PTP PLA complexes per cell. Mean±s.d., n = 30 cells per condition. * P <0.05, t -test. ( f ) Quantification of VEGFR2/VE-PTP PLA complexes at junctions per cell. Mean±s.d., n =45 cells per condition repeated twice. ** P <0.01, *** P <0.001, t -test. ( g ) VEGFR2/VE-PTP PLA was performed in HUVECs transfected with control and tie2 siRNA and treated with VEGF and Ang1 as in d , followed by PLA for VEGFR2/VE-PTP complexes, and immunostaining with anti-ZO1 antibodies. Scale bars, 20 μm. High-magnification insets show PLA products at junction; scale bars, 10 μm. ( h ) Quantification of VEGFR2/VE-PTP PLA complexes per cell. Mean±s.d., n = 100 cells per condition, repeated twice. ** P <0.01, t -test.

Article Snippet: Antibodies for immunofluorescence: anti-mouse CD31 (BD Biosciences), anti-mouse NG2 (Millipore), anti-mouse Podocalyxin (R&D Systems), anti-mouse Moesin (Abcam), anti-mouse VE-cadherin (BD Biosciences), anti-VE-PTP (hPTPb1-8) (ref. ), anti-mouse VEGFR2 (R&D Systems), anti-human VEGFR2 (R&D Systems), anti-human pY1175 VEGFR2 (Cell Signaling), anti-human Tie2 (R&D Systems), anti-VE-cadherin (BD Biosciences) and anti-pY658 VE-cadherin .

Techniques: Transfection, Control, Immunostaining

Journal: Nature Communications

Article Title: VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation

doi: 10.1038/ncomms2683

Figure Lengend Snippet: ( a ) WT EBs in 3D collagen with VEGF (20 ng ml −1 ) in the presence or absence of Tie2/Fc protein (4 μg ml −1 ) show CD31 (green), podocalyxin (red) and Hoechst 33342 (blue). Dashed lines show position of vertical-view images displayed in panels labelled tip, stalk and root. Scale bars, 10 μm. Asterisk, lumen; arrowhead, abnormal podocalyxin distribution. ( b ) Quantification of ECs with abnormal podocalyxin distribution. Mean±s.d., n = 20 sprouts per condition. *** P <0.001, t -test. ( c ) pVEGFR2 (green), VEGFR2 (red) and CD31 (white) at day 14 in WT EBs treated or not with Tie2/Fc protein (4 μg ml −1 ). Arrows indicate pVEGFR2. Scale bars; 20 μm. ( d ) Ratio of pVEGFR2/VEGFR2 intensities from sprout tip to stalk. Data show the mean; n = 8 sprouts/condition. ( e ) Ang1 transcript expression/HPRT expression in WT and ve-ptp −/− EBs in 2D collagen with VEGF (20 ng ml −1 ) at day 14. Mean±s.d. Statistics are based on two independent experiments with n =150 EBs per experiment.

Article Snippet: Antibodies for immunofluorescence: anti-mouse CD31 (BD Biosciences), anti-mouse NG2 (Millipore), anti-mouse Podocalyxin (R&D Systems), anti-mouse Moesin (Abcam), anti-mouse VE-cadherin (BD Biosciences), anti-VE-PTP (hPTPb1-8) (ref. ), anti-mouse VEGFR2 (R&D Systems), anti-human VEGFR2 (R&D Systems), anti-human pY1175 VEGFR2 (Cell Signaling), anti-human Tie2 (R&D Systems), anti-VE-cadherin (BD Biosciences) and anti-pY658 VE-cadherin .

Techniques: Expressing

Journal: Nature Communications

Article Title: VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation

doi: 10.1038/ncomms2683

Figure Lengend Snippet: ( a ) WT and ve-ptp −/− teratoma sections show CD31 (green), podocalyxin (red) and Hoechst 33342 (blue). Asterisk, lumen. Scale bars, 20 μm. ( b ) Podocalyxin intensity/CD31-positive area (mm 3 ) in a . Mean±s.d.; n = 15 CD31-positive vessels of 50 μm length/3 teratomas/genotype. * P <0.05, t -test. ( c ) WT and ve-ptp −/− teratoma sections. Left: 16-colour intensity scale representation of pVEGFR2 immunostaining. Right: Hoechst 33342 (blue), pY1175 VEGFR2 (green), and CD31 (grey). Scale bars, 20 μm. ( d ) pY1175 VEGFR2 intensity/CD31-positive area (mm 2 ) in c . Mean±s.d.; n = 12 (WT) and 15 ( ve-ptp −/− ) blood vessels of 30 μm length from 3 teratomas per genotype. ** P <0.01, t -test. ( e ) Lectin-perfused WT and ve-ptp −/− teratomas. Hoechst 33342 (blue), lectin-FITC (green), pY1175 VEGFR2 (red) and VE-PTP (white). Right: control immunostaining without primary antibodies. Asterisk, lumen. Arrows show VE-PTP expression in WT teratomas. Arrowheads show pVEGFR2 in ve-ptp −/− teratomas. Scale bars, 20 μm. ( f ) Lectin-perfused WT and ve-ptp −/− teratomas show lectin-FITC (green), VE-cadherin (red) and VE-PTP (white). Asterisk, lumen. Arrowheads in the lower ve-ptp −/− panel indicate fragmented VE-cadherin immunostaining. Scale bars, 20 μm. ( g ) Lectin-perfused B16 F10 mouse melanomas show lectin-FITC (green), VE-cadherin (VE-cad; red) and VE-PTP (white). Asterisk, lumen. Arrowheads in the lower panel indicate fragmented VE-cadherin immunostaining. Scale bars, 20 μm. ( h ) Model illustrating the contribution of VE-PTP in silencing VEGFR2 and Tie2 at junctions to support proper EC polarity and vessel morphogenesis. VE-PTP exists in complex with VEGFR2 and Tie2 in the WT condition (left). VEGF induces activation of VEGFR2 and in parallel, dissociation from VE-PTP. VEGF and Ang1 induce translocation of the trimeric complex to junctions where the activated receptors are silenced by VE-PTP. This is compatible with formation of polarized and lumenized vessels. In the VE-PTP-deficient condition ( ve-ptp −/− ; right), VEGF and Ang1 induce activation and translocation of receptors to junctions, where excess activity leads to VE-cadherin phosphorylation and formation of unpolarized and lumen-less pathological vasculature.

Article Snippet: Antibodies for immunofluorescence: anti-mouse CD31 (BD Biosciences), anti-mouse NG2 (Millipore), anti-mouse Podocalyxin (R&D Systems), anti-mouse Moesin (Abcam), anti-mouse VE-cadherin (BD Biosciences), anti-VE-PTP (hPTPb1-8) (ref. ), anti-mouse VEGFR2 (R&D Systems), anti-human VEGFR2 (R&D Systems), anti-human pY1175 VEGFR2 (Cell Signaling), anti-human Tie2 (R&D Systems), anti-VE-cadherin (BD Biosciences) and anti-pY658 VE-cadherin .

Techniques: Immunostaining, Control, Expressing, Activation Assay, Translocation Assay, Activity Assay, Phospho-proteomics

Journal: Journal of Oncology

Article Title: Anoikis Resistance as a Further Trait of Acidic-Adapted Melanoma Cells

doi: 10.1155/2019/8340926

Figure Lengend Snippet: An anoikis resistant-like phenotype in acidic melanoma cells. (a) Representative images of western blot for N-cad, EGFR, pAKT/AKT, pERK/ERK, Tie1, IKB, and β -actin of A375M6 melanoma cells exposed to standard medium (pH 7.4), to an acidified medium for 24 hours (transient exposure, pH 6.7) or to a reduced pH medium, for approximately three months (chronic exposure, pH 6.7c), and (right) densitometry graph of protein expression. (b) Representative images (left) of invasiveness of melanoma cells grown in different pH conditions in the presence or absence of 25 μ M Ilomastat (a metalloproteinases inhibitor) and quantitative analysis (right) of the number of cells that migrated through Matrigel. (c) Representative images (left) of flow cytometric analysis of α v β 3 and of α v β 5 integrin expression of A375M6 melanoma cells grown in different pH conditions and (right) quantitative analysis of integrin expression as percentage of increment in Mean Fluorescent Intensity. Each experiment was conducted in triplicate and data are expressed as mean ± SEM of at least three independent experiments. ∗ p<0.05.

Article Snippet: The primary antibodies were as follows: rabbit anti-EGFR (1:500 Cell Signaling Technology, Danvers, MA, USA), rabbit anti-N-Cadherin (1:1000 Biorbyt, Cambridge, UK), mouse anti-cleaved PARP 1 (1:200 Santa Cruz Biotechnology, Santa Cruz, California), rabbit anti-p-AKT (Cell Signaling Technology, Danvers, MA, US), rabbit anti-AKT (1:1000 Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pERK (1:1000 Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ERK (1:1000 Cell Signaling Technology, Danvers, MA, USA), mouse anti-tie1 (1:200 Santa Cruz Biotechnology, Santa Cruz, California), mouse anti-IKB alpha (1:500 GeneTex, Irvine, CA USA), and mouse anti- β actin monoclonal antibody (1:2000, GeneTex, Irvine, CA,USA).

Techniques: Western Blot, Expressing

Journal: Nature cardiovascular research

Article Title: Angiopoietin-TIE2 feedforward circuit promotes PIK3CA-driven venous malformations.

doi: 10.1038/s44161-025-00655-9

Figure Lengend Snippet: Fig. 6 | Increased TIE2 phosphorylation and SMC coverage in Pik3ca-driven VM in mice. a, PLA staining of activated TIE2 on ear skin paraffin sections from Pik3caH1047R;Vegfr1-CreERT2 and Cre− littermate Ctrl mice, detected using pTyr and TIE2 antibodies. DAPI marks cell nuclei. b, Quantification of PLA signals within PECAM1+ blood vessels. Data represent the number of PLA dots per μm2 of PECAM1+ vessel area, mean ± s.d. (n = 8 (Ctrl) and n = 26 (Pik3caH1047R) vessels from four mice per genotype, unpaired two-tailed Student’s t-test, ****P = 0.0000054). c, Immunofluorescence of ear skin paraffin sections from Pik3caH1047R;Vegfr1- CreERT2 and Cre− littermate Ctrl mice using phospho-TIE2 antibodies. d, Phospho- TIE2 signal within EMCN+ vessels, represented as corrected total cell fluorescence (CTFC) of EMCN+ vessel area. Data points represent pTIE2 CTFC, mean ± s.d. (n = 20 (Ctrl) and n = 31 (Pik3caH1047R) vessels from two mice per genotype, unpaired two-tailed Student’s t-test, ****P = 0.000096; Extended Data Fig. 8d). e, Whole-mount immunofluorescence of ear skin from Pik3caH1047R;

Article Snippet: Anti-TIE2 antibody (goat, AF313) was obtained from R&D Systems and used at a dilution of 1:1,000.

Techniques: Phospho-proteomics, Staining, Two Tailed Test, Immunofluorescence, Fluorescence

Journal: Nature cardiovascular research

Article Title: Angiopoietin-TIE2 feedforward circuit promotes PIK3CA-driven venous malformations.

doi: 10.1038/s44161-025-00655-9

Figure Lengend Snippet: Fig. 7 | Increased TIE2 phosphorylation in human PIK3CA-driven VMs. a, H&E-stained paraffin sections of cutaneous VMs from individuals with PIK3CA (top) or TEK (below) mutations. Areas defined as non-lesional (NL) and VM lesions are indicated. b, PLA staining of activated TIE2, detected using pTyr and TIE2 antibodies, in representative vessels from indicated NL and VM regions. DAPI marks cell nuclei. c, Quantification of PLA signals within PECAM1+ veins, represented as mean PLA dots per EC nucleus ± s.d. (n = 7 (PIK3CA), n = 6 (TEK), n = 2 (Ctrl) and n = 5 (NL) individuals, with symbols indicating different mutations; ordinary one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test, **P(PIK3CA VM versus Ctrl vein) = 0.0027 and **P(TEK VM versus Ctrl vein) = 0.0078; Extended Data Fig. 7). d, Representative immunofluorescence image (left) and quantification (right) showing the

Article Snippet: Anti-TIE2 antibody (goat, AF313) was obtained from R&D Systems and used at a dilution of 1:1,000.

Techniques: Phospho-proteomics, Staining, Comparison, Immunofluorescence