Journal: Science signaling

Article Title: Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

doi: 10.1126/scisignal.aad3812

Figure Lengend Snippet: Fig. 1. VEGF-A165, a ligand-blocking anti-NRP1, and a tetrameric CendR peptide induce NRP1 accumula- tion at endothelial cell–cell contacts. (A to D) Epifluorescence microscope images of permeabilized HUVEC monolayers. (A) Cells were stimulated with VEGF-A165 and stained with an antibody against NRP1 (red). (B) After incubation with the ligand-blocking anti-NRP1 antibody (anti-NRP1) (lower panel) or its cor- responding control sheep IgG (upper panel), cells were stimulated with VEGF-A165. HUVECs were stained with an antibody specific for NRP1 (red) and secondary anti-sheep antibody (green). Accumulation of NRP1 at cell-cell contacts was observed in the absence of VEGF-A165 (white arrows). (C) Cells were stimulated with anti-NRP1 and stained with secondary anti-sheep antibody (green). (D) Cells were stimulated with NA-RPARPAR peptide and stained with an antibody specific for NRP1 (red). Nuclei were stained with Hoechst (blue). Images in (A) to (D) are representative of n > 3 independent experiments. Scale bars, 20 mm.

Article Snippet: The ligand-blocking sheep anti-human NRP1 and goat anti-rat NRP1 antibodies were purchased from R&D Systems.

Techniques: Blocking Assay, Microscopy, Staining, Incubation, Control

Journal: Science signaling

Article Title: Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

doi: 10.1126/scisignal.aad3812

Figure Lengend Snippet: Fig. 2. VEGF-A165, NA-RPARPAR, and anti-NRP1 induce NRP1 junctional localization and endothelial leakage in vitro and in vivo. (A) Confocal microscopy images of permeabilized HUVEC monolayers stimu- lated with VEGF-A165, NA-RPARPAR, and anti-NRP1 and stained with an antibody against VE-cadherin (green). NRP1 (red) was stained with an antibody against NRP1 (upper and middle panels) or directly with the secondary anti-sheep antibody (lower panel). Nuclei were stained with Hoechst (blue). White arrows indicate colocalization between NRP1 and VE-cadherin. Images are representative of four independent experiments. Scale bar, 20 mm. (B) HUVEC monolayers were seeded on top of Transwell filters and stimulated with PBS, VEGF-A165 (VEGF), NA-RPARPAR (NA-R), and anti-NRP1 (Ab). Leakage of FITC- dextran 70 kD from the upper to the lower well was measured by comparing fluorescence values at 520 nm (mean ± SEM; n = 9 independent experiments; the median from three to six replicates per independent experiment was used for statistical analyses; Friedman test followed by Dunn’s multiple com- parison post hoc test; *P < 0.05 and **P < 0.01). (C) Wild-type mice were systemically injected with Evans Blue and then with PBS, VEGF-A165, NA-RPARPAR, anti-NRP1 antibody, and their respective controls. The extravasated dye concentration was measured at 620 nm, and results were expressed as a ratio between the tested substance and its control (mean; n = 10 to 15 mice per treatment; Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test; **P < 0.01 and ***P < 0.001).

Article Snippet: The ligand-blocking sheep anti-human NRP1 and goat anti-rat NRP1 antibodies were purchased from R&D Systems.

Techniques: In Vitro, In Vivo, Confocal Microscopy, Staining, Fluorescence, Injection, Concentration Assay, Control, Comparison

Journal: Science signaling

Article Title: Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

doi: 10.1126/scisignal.aad3812

Figure Lengend Snippet: Fig. 3. Unlike VEGF-A165, NA-RPARPAR and anti-NRP1 do not activate VEGFR-2, Akt, p38, ERK, or FAK. (A to C) HUVEC monolayers were stimu- lated with VEGF-A165, NA-RPARPAR, or anti-NRP1 antibody. (A) Cell ly- sates were immunoprecipitated with an antibody against VEGFR-2 and blotted with anti–VEGFR-2 and anti-phosphotyrosine (pY) antibodies. The corresponding total lysates were blotted for VEGFR-2 (n = 4 independent experiments). (B and C) Blotting was performed on total lysates with anti- bodies against pAkt, pERK1/2, p-p38 (n = 3 independent experiments) (B),

Article Snippet: The ligand-blocking sheep anti-human NRP1 and goat anti-rat NRP1 antibodies were purchased from R&D Systems.

Techniques: Immunoprecipitation

Journal: Science signaling

Article Title: Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

doi: 10.1126/scisignal.aad3812

Figure Lengend Snippet: Fig. 4. VEGF-A165, NA-RPARPAR, and anti-NRP1 induce NRP1 relocalization and vascular leakage inde- pendently of VEGFR-2 activation. (A and B) HUVEC monolayers were treated with PTK/ZK or dimethyl sulfoxide (DMSO) before stimulation. ct, control. (A) Cell lysates were immunoprecipitated (IP) with VEGFR-2 antibody. Immunoprecipitates were blotted for VEGFR-2 and phosphotyrosine, and corresponding total lysates for VEGFR-2 (R2). Representative scans of five experiments. (B) HUVEC mono- layers were stained for NRP1 (red), nuclei were stained with Hoechst (blue), and cells were imaged with an epifluorescence microscope. Representative images from three experiments. Scale bar, 25 mm. (C and D) HUVECs were transfected with noncoding (NC) small interfering RNA (siRNA) or siRNAs coding for VEGFR-2 (KDR) (siRNA 1 and siRNA 2). (C) KDR mRNA relative expression was quantified by quantita- tive real-time polymerase chain reaction (qRT-PCR) (mean ± SEM; n = 3 independent experiments). (D) Epifluorescence images of transfected HUVECs in (C), stimulated with VEGF-A165, NA-RPARPAR, or anti- NRP1. Cells were stained for NRP1 (red), and nuclei were stained with Hoechst (blue). Representative images of three experiments. Scale bar, 20 mm. (E to H) Mice were injected intraperitoneally with DMSO or PTK/ZK before systemic treatment with Evans Blue. Leakage was induced with VEGF-A165 (E), NA-RPARPAR (F), anti-NRP1 (G), or VEGF-A121 (H), and results were expressed as a ratio between the tested substance and its respective control (mean; n = 10 to 20 mice per condition; Mann-Whitney test).

Article Snippet: The ligand-blocking sheep anti-human NRP1 and goat anti-rat NRP1 antibodies were purchased from R&D Systems.

Techniques: Activation Assay, Control, Immunoprecipitation, Staining, Microscopy, Transfection, Small Interfering RNA, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Injection, MANN-WHITNEY

Journal: Science signaling

Article Title: Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation.

doi: 10.1126/scisignal.aad3812

Figure Lengend Snippet: Fig. 5. The NRP1 cytoplasmic domain mediates vascular leakage. (A and B) HUVECs overexpressing GFP, full-length NRP1, or NRP1 deprived from the cytoplasmic domain (NRP1DC) were stimulated with VEGF-A165. (A) Cell lysates were immunoprecipitated with VEGFR-2 antibody. Immunopre- cipitates were blotted for VEGFR-2 and phosphotyrosine, and total lysates were blotted for NRP1 and actin (n = 3 independent experiments). (B) HUVECs overexpressing GFP, NRP1, or NRP1DC were stained with anti- NRP1 (red). Nuclei were stained with Hoechst (blue). Representative images of three independent experiments. Scale bar, 20 mm. (C to E) Wild-type mice (NRP1cyto+/+) and mice expressing cytoplasmatically trun-

Article Snippet: The ligand-blocking sheep anti-human NRP1 and goat anti-rat NRP1 antibodies were purchased from R&D Systems.

Techniques: Immunoprecipitation, Staining, Expressing

Journal: Breast Cancer Research : BCR

Article Title: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

doi: 10.1186/s13058-022-01501-7

Figure Lengend Snippet: High NRP1 expression predicts shorter time to relapse- and distant metastasis-free survival in ER-negative breast cancer patient cohorts. a Association of NRP1 expression quartiles (Q1–Q4) with overall survival in the BrCa TCGA cohort . Data were obtained from UCSC Xena . KM Plotter analysis of relapse-free survival (left panel; RFS) and distant metastasis-free survival (right panel; DMFS) in an b unselected patient cohort (RFS; n = 4929, DMFS; n = 2765; months survival displayed as median survival), c ER-positive only (RFS; n = 3768, DMFS; n = 2016; months survival displayed as median survival) and d ER-negative only (RFS; n = 1161, DMFS; n = 749; months survival displayed as upper quartile survival) tumor subcohorts [ , ]

Article Snippet: Anti-human APC-conjugated NRP1 antibody (R&D System, FAB3870A) or IgG-APC control (R&D System, IC003A) was added and cells incubated on ice for 45 min.

Techniques: Expressing

Journal: Breast Cancer Research : BCR

Article Title: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

doi: 10.1186/s13058-022-01501-7

Figure Lengend Snippet: NRP1 is over-expressed in the claudin-low molecular subtype of breast cancer. a Heatmap showing NRP1 expression association with PAM50, claudin-low, core claudin-low (CoreCL), ER and HER2 tumor status, as well as core claudin-low signature genes. b NRP1 mRNA expression (log2 signal) in intrinsic breast cancer subtypes and claudin-low tumors (CLDN low ) in the METABRIC patient dataset ( n = 1904), obtained through cBioPortal . c NRP1 mRNA expression across intrinsic subtypes subdivided into claudin-low (CL) and non-claudin-low tumors. Correlation of claudin-low di up-gene (CLDN low UP GES) and dii down-gene (CLDN low DN GES) GSVA-derived signature scores with NRP1 expression. Claudin-low gene signature scores were obtained via GSVA. Sample subtype is represented according to color scheme used in A-C. e NRP1 mRNA expression in METABRIC core claudin-low (CoreCL), non-core claudin-low (OtherCL) and non-claudin-low tumors . Error bars represent SEM, * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001

Article Snippet: Anti-human APC-conjugated NRP1 antibody (R&D System, FAB3870A) or IgG-APC control (R&D System, IC003A) was added and cells incubated on ice for 45 min.

Techniques: Expressing, Derivative Assay

Journal: Breast Cancer Research : BCR

Article Title: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

doi: 10.1186/s13058-022-01501-7

Figure Lengend Snippet: NRP1 expression is associated with in vivo tumor progression, cancer stemness and spheroid-initiating potential. a Western blot analysis of NRP1 expression in non-targeting control (shNT) and NRP1 shRNA-silenced (shNRP1 ) SUM159 cells inoculated into mice. b Post-inoculation tumor volumes, c tumor volume at week 8 post-inoculation and d Kaplan–Meier analysis of overall survival in SUM159 shNT and NRP1 knockdown groups. e Representative images of NRP1 immunohistochemistry in shNT and shNRP1 tumors and f quantification of NRP1 IHC staining across shNT and NRP1 knockdown groups. g NRP1 expression across CL1, CL2 and CL3 claudin-low subtypes as well PAM50 classifiers in the METABRIC dataset obtained via cBioportal [ , ]. h qPCR (left and center panel; n = 3) and Western blot (right panel) analysis of ZEB1 expression in HS578T cells after 72 h NRP1 knockdown versus NT control. i qPCR analysis of ITGA6 mRNA expression in HS578T cells (leftmost panel; NRP1 expression shown in h ) and SUM159 cells (center and right panel) after 72 h NRP1 knockdown versus NT control ( n = 3). j Western blot showing ITGA6 expression in HS578T, SUM159 and MDA-MB-231 cells after 72 h NRP1 knockdown versus NT control. k Western blot showing expression of ZEB1 and NRP1 in FACS sorted CD44 + /CD24 lo and CD44 + /CD24 hi populations of SUM159 cells. l Number of spheroids (> 50 µM) formed by day 6 following seeding of single cell SUM159 and Hs578T cell cultures containing 1,200 cells in the presence of 50 µg/ml Vesencumab (red lines) or IgG control (black lines). n = 5, along with m representative images of ( mi ) SUM159 and ( mii ) Hs578T spheroid cultures at days 4 and 6 post-seeding. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, **** p ≤ 0.0001, error bars represent SEM

Article Snippet: Anti-human APC-conjugated NRP1 antibody (R&D System, FAB3870A) or IgG-APC control (R&D System, IC003A) was added and cells incubated on ice for 45 min.

Techniques: Expressing, In Vivo, Western Blot, Control, shRNA, Knockdown, Immunohistochemistry

Journal: Breast Cancer Research : BCR

Article Title: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

doi: 10.1186/s13058-022-01501-7

Figure Lengend Snippet: NRP1 inhibition suppresses in vivo growth of claudin-low SUM159 orthotopic xenografts. a Luciferase-derived luminescence signal from SUM159 luc primary tumors as imaged by the IVIS Spectrum In Vivo Imaging System at 7 weeks post-tumor inoculation. Endpoint (7 weeks) mean b tumor luciferase intensity, c tumor weight and d tumor volume. e Tumor growth curves in IgG control and Vesencumab groups. f Endpoint (7 weeks) tumors from IgG control and Vesencumab groups; two additional tumors from the Vesencumab-treated group were too small to collect. g Representative images of H&E staining and Ki67 and CD31 immunohistochemistry of Vesencumab and IgG control treated tumors, with quantification of hi Ki67 and hii CD31 staining across all tumors. ‘Ves’; Vesencumab. Error bars represent SEM; ** p < 0.001; **** p < 0.0001. For a – e , n = 12–14. For g – h , n = 12

Article Snippet: Anti-human APC-conjugated NRP1 antibody (R&D System, FAB3870A) or IgG-APC control (R&D System, IC003A) was added and cells incubated on ice for 45 min.

Techniques: Inhibition, In Vivo, Luciferase, Derivative Assay, In Vivo Imaging, Control, Staining, Immunohistochemistry

Journal: Breast Cancer Research : BCR

Article Title: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

doi: 10.1186/s13058-022-01501-7

Figure Lengend Snippet: NRP1 inhibition suppresses EGFR and PDGFRα signaling in claudin-low cells. ai Receptor tyrosine kinase array showing EGFR and PDGFRα expression in SUM159 cells after 60 min treatment with 50 μg/mL IgG or Vesencumab (top panel), or 72 h after transfection with NRP1 targeting siRNA (siNRP1 or siNRP1 ) versus non-targeting (NT) control (bottom panel), with aii corresponding densitometry. Ref1 and Ref2 represent positive controls. b Western blot showing NRP1, total EGFR, phospho-EGFR (pEGFR Y1068), total PDGFRα and phospho-PDGFRα (Y1018) expression in SUM159, MDA-MB-231 and HS578T cells after 72 h NRP1 knockdown versus NT control. c Correlation analysis between NRP1 and EGFR (right panel; Pearson: 0.24, p = 2.7e−26) and PDGFRα (left panel; Pearson: 0.52, p = 3.47e−130) mRNA expression in the METABRIC dataset ( n = 1904) . Data was obtained from cBioportal. d Immunohistochemical analysis of phosphorylated (T202/Y204) p42/44 levels in endpoint (7 weeks post-tumor inoculation) Vesencumab or IgG control treated tumors, with e representative images (right panel); scale bars = 50 µm, n = 5–6. Error bars represent SEM, * p ≤ 0.05; ** p ≤ 0.01 versus control

Article Snippet: Anti-human APC-conjugated NRP1 antibody (R&D System, FAB3870A) or IgG-APC control (R&D System, IC003A) was added and cells incubated on ice for 45 min.

Techniques: Inhibition, Expressing, Transfection, Control, Western Blot, Knockdown, Immunohistochemical staining

Journal: The Journal of Experimental Medicine

Article Title: VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation

doi: 10.1084/jem.20160311

Figure Lengend Snippet: NRP1 localization in adult vascular endothelium. Whole-mount immunostaining of adult mouse ear dermis for NRP1, PECAM1, and SMA (A) and adult mouse retina for NRP1, PECAM1, and CDH5 (B), including control staining for the secondary antibodies used to detect NRP1 (anti–goat) and CDH5 (anti–rabbit), together with the primary and secondary antibody for PECAM1 (three independent experiments). Arrowheads indicate examples of endothelial junctions sites enriched for NRP1 in venules. a, artery; v, vein. (C) Single optical sections from the boxed areas in A and C’ and B and C’’ were analyzed for pixel intensity along a virtual line crossing the blood vessel. Bars, 50 µm.

Article Snippet: HDMEC and MLEC were fixed in 4% formaldehyde in PBS for 15 min. To detect NRP1 in HDMEC, we used mouse anti–human NRP1 (R&D Systems).

Techniques: Immunostaining, Control, Staining

Journal: The Journal of Experimental Medicine

Article Title: VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation

doi: 10.1084/jem.20160311

Figure Lengend Snippet: VEGF164-induced vascular leakage depends on VEGFR2, NRP1 and VEGF-binding to NRP1. (A) Evans Blue leaks from the circulation into the dermis after intradermal injection of VEGF164, but not PBS; the circles indicate the tissue area around the injection sites that was excised for dye extraction. Bar, 1 cm. (B and C) Vegfr2 fl/fl (B) and Nrp1 fl/fl (C) mice expressing or lacking the endothelial Cdh5-CreERT2 transgene were tamoxifen treated to induce gene deletion; immunoblotting of liver (B) or skin (C) lysates with the indicated antibodies (left) confirmed gene deletion, whereas Miles assays with PBS versus VEGF164 (right) showed defective VEGF164-induced leakage. (D) Schematic representation of NRP1 mutants with defective VEGF164 binding to NRP1. (E–G) Miles assay with PBS versus VEGF164 in mutant and wild-type littermates of the indicated genotypes. Immunoblotting of skin lysates with the indicated antibodies (G) showed normal NRP1 levels in Nrp1 D320K/D320K mice compared with littermate controls. In B–G, leakage was measured as optical density and expressed as fold change relative to PBS, mean ± SEM; n = 5 each (B and G), n = 5 controls, n = 6 mutants (C), n = 8 controls, n = 10 mutants (E), n = 4 controls, n = 7 mutants (F); asterisks indicate significant P-values for permeability-inducing agents versus PBS: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant, P > 0.05; paired Student’s t test. Hash tags indicate significant P-values for permeability-induction in mutants versus controls ( # , P < 0.05; ## , P < 0.01; ns, P > 0.05; unpaired Student’s t test).

Article Snippet: HDMEC and MLEC were fixed in 4% formaldehyde in PBS for 15 min. To detect NRP1 in HDMEC, we used mouse anti–human NRP1 (R&D Systems).

Techniques: Binding Assay, Injection, Extraction, Expressing, Western Blot, Mutagenesis, Permeability

Journal: The Journal of Experimental Medicine

Article Title: VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation

doi: 10.1084/jem.20160311

Figure Lengend Snippet: The NCD is required to mediate VEGF164-induced vascular leakage, but does not regulate baseline vascular permeability. (A) Schematic representation of mutations that impair NRP1 intracellular activity. (B–E) Miles assay with the indicated substances in mutant and wild-type littermates of the indicated genotypes; leakage was measured as optical density and expressed as fold change relative to PBS, mean ± SEM; n = 6 controls, n = 5 mutants (B), n = 7 each (C), n = 5 each (D), n = 4 each (E); asterisks indicate significant P-values for permeability-inducing agents versus PBS (*, P < 0.05; **, P < 0.01; ***, P < 0.001; paired Student’s t test), and hash tags indicate significant P-values for permeability-induction in mutants versus controls ( ## , P < 0.01; ns, not significant, P > 0.05; unpaired Student's t test). (F) Evans Blue content in the indicated organs 24 h after systemic injection in Nrp1 cyto/cyto mice and wild-type littermates. Values are normalized to tissue weight and Evans Blue levels in the blood; mean ± SEM; n = 3 each; ns, P > 0.05; unpaired Student’s t test.

Article Snippet: HDMEC and MLEC were fixed in 4% formaldehyde in PBS for 15 min. To detect NRP1 in HDMEC, we used mouse anti–human NRP1 (R&D Systems).

Techniques: Permeability, Activity Assay, Mutagenesis, Injection

Journal: The Journal of Experimental Medicine

Article Title: VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation

doi: 10.1084/jem.20160311

Figure Lengend Snippet: NRP1 loss impairs VEGF165-induced SFK activation in human ECs . (A and B) Immunostaining of confluent HDMEC cultures in growth medium under nonpermeabilizing conditions with an antibody specific for human NRP1 (A) or under permeabilizing conditions with antibodies for CDH5 and NRP1 together with DAPI to visualize cell nuclei (B); three independent experiments. Single channels in B are shown separately in grayscale, and the boxed area is shown in higher magnification on the right. Bar, 50 µm. (C) Immunostaining of confluent HDMEC cultures under permeabilizing conditions for pSFK together with CDH5 and DAPI after serum withdrawal, followed by stimulation with VEGF165 for the indicated times (three independent experiments). The corresponding single pSFK channels are shown beneath each panel in grayscale as well as the rainbow pixel intensity scale. Bar, 50 µm. (D–F) Confluent HDMEC cultures transfected with si-control or siNRP1 were serum-starved and treated with VEGF165 for the indicated times. Lysates were used for immunoblotting with the indicated antibodies (D), followed by quantification of pVEGFR2 (Y1175; E) and pSFK (F) induction relative to tVEGFR2 and GAPDH, respectively. Each of the two vertical lines indicates a group of immunoblots from a single gel, with both gels containing aliquots of the same protein lysate. Data for si-control and siNRP1-treated cells are expressed as ratio (E) or fold change, for VEGF165 treatment at different time points relative to 0 min (F) mean ± SEM; n = 4 independent experiments; asterisks indicate significant P-values for pSFK induction after VEGF165 treatment (*, P < 0.05; **, P < 0.01; paired Student’s t test). Hash tags indicate significant P-values for reduced pVEGFR2 and pSFK levels in siNRP1 versus si-control at the corresponding time points ( ## , P < 0.01; unpaired Student’s t test).

Article Snippet: HDMEC and MLEC were fixed in 4% formaldehyde in PBS for 15 min. To detect NRP1 in HDMEC, we used mouse anti–human NRP1 (R&D Systems).

Techniques: Activation Assay, Immunostaining, Transfection, Control, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation

doi: 10.1084/jem.20160311

Figure Lengend Snippet: VEGFR2 and NRP1 are required for VEGF165-induced SFK activation via ABL1 . (A–C) Confluent HDMEC cultures transfected with si-control or siNRP1 were serum-starved and treated with VEGF165 for the indicated times. Cultures were also treated with vehicle (-) or PTK/ZK (+) for 30 min before VEGF165 stimulation. Lysates were used for immunoblotting with the indicated antibodies (A), followed by quantification of pSFK levels (B, left) and pCRKL levels (B’, right, and C) relative to GAPDH (four independent experiments). Each of the two vertical lines indicated a group of immunoblots from a single gel, with both gels containing aliquots of the same protein lysate. (D–E) Confluent HDMEC cultures transfected with si-control or siABL1 were serum-starved and treated with VEGF165 for the indicated times. Lysates were used for immunoblotting with the indicated antibodies (D), followed by quantification of pSFK levels (E, left) and pCRKL levels (E’, right) relative to GAPDH (four independent experiments). Each of the two vertical lines indicates a group of immunoblots from a single gel, with both gels containing aliquots of the same protein lysate. The spacer line (D, bottom) separates lanes 4–6 (left) from lanes 1–3 (right) of immunoblots from the gel in Fig. S1. (F–G) Confluent HDMEC cultures were serum-starved and treated with vehicle, Imatinib or PP2 for 30 min before VEGF165 stimulation for the indicated times. Lysates were used for immunoblotting with the indicated antibodies (F), followed by quantification of pSFK levels (G, left) and pCRKL levels (G’, right) relative to GAPDH (three independent experiments). Each of the two vertical lines indicates a group of immunoblots obtained from a single gel, with both gels containing aliquots of the same protein lysate. In B, E, and G (left) data are expressed as fold change, mean ± SEM, in VEGF165-treated cells at 5 and 15 min relative to 0 min; in C and B, E, and G (right), data are expressed as fold change, mean ± SEM, in VEGF165-treated cells at 5 and 15 min relative to control cells at 0 min; asterisks indicate P-values for induction after VEGF165 treatment (*, P < 0.05; **, P < 0.01; ***, P < 0.001; paired Student’s t test); hash tags indicate significant P-values for different treatments at corresponding time points ( # , P < 0.05; ## , P < 0.01; ### , P < 0.001; unpaired Student’s t test; n ≥ 3 independent experiments). (H) Confluent HDMEC cultures transfected with si-control or siNRP1 and serum-starved were treated with VEGF165 for the indicated times and lysates used for immunoblotting with the indicated antibodies (three independent experiments). (I) Model for VEGF165-induced vascular permeability signaling including the point of interference by pharmacological inhibitors used in this study.

Article Snippet: HDMEC and MLEC were fixed in 4% formaldehyde in PBS for 15 min. To detect NRP1 in HDMEC, we used mouse anti–human NRP1 (R&D Systems).

Techniques: Activation Assay, Transfection, Control, Western Blot, Permeability

Journal: The Journal of Experimental Medicine

Article Title: VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation

doi: 10.1084/jem.20160311

Figure Lengend Snippet: NCD loss impairs VEGF164-induced SFK activation in the mouse . (A) Nrp1 cyto/cyto and wild-type ears were injected with VEGF164 or PBS for 20 min and lysates used for immunoblotting with the indicated antibodies (two independent experiments). (B) Confluent MBECs from Nrp1 cyto/cyto and wild-type brains were serum-starved and treated with VEGF164 for the indicated time points. Lysates were used for immunoblotting with the indicated antibodies (left), followed by quantification of pSFK relative to GAPDH levels (right). Data are expressed as fold change, mean ± SEM, in VEGF164-treated cells at 10 and 20 min relative to 0 min; n = 3 independent experiments; asterisk indicates significant P-value for induction after VEGF164 treatment (*, P < 0.05; paired Student’s t test); hash tag indicates significant P-value for different genotypes at corresponding time point ( # , P < 0.05; unpaired Student’s t test). (C–E) Confluent MLECs from Nrp1 cyto/cyto and wild-type lungs were serum-starved and treated with VEGF164 for the indicated times and immunostained under permeabilizing conditions using an antibody for pSFK (C) or lysed for immunoblotting with the indicated antibodies (D and E); cells were counterstained with DAPI (two independent experiments each). Bar, 50 µm.

Article Snippet: HDMEC and MLEC were fixed in 4% formaldehyde in PBS for 15 min. To detect NRP1 in HDMEC, we used mouse anti–human NRP1 (R&D Systems).

Techniques: Activation Assay, Injection, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation

doi: 10.1084/jem.20160311

Figure Lengend Snippet: The NCD promotes vascular leakage, but not neovascularisation, in a mouse model of CNV. (A) Adult eye sections immunostained for NRP1 and CDH5; nuclei were counterstained with DAPI (two independent experiments); NRP1 staining is shown separately on the right. An extension of the squared area is shown at higher magnification in (A’–A’’’); the NRP1 channel is shown separately in (A’’); the DIC image is superimposed in A’’’. RGC, retinal ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. (B) Evans Blue content in the indicated ocular tissues in Nrp1 cyto/cyto mice and wild-type littermates; mean ± SEM; n = 3 mice; ns, P > 0.05; unpaired Student's t test. (C) ELISA shows that VEGF is up-regulated in the RPE/choroid of wild-type mice on D3 after laser injury in the CNV model ( n = 4) compared with eyes before laser injury ( n = 6); data are expressed as mean ± SEM; the asterisk indicates a significant increase in VEGF levels on D3 (***, P < 0.001; unpaired Student’s t test). (D) Pathological vascular leakage in Nrp1 cyto/cyto mice and wild-type littermates. On D3 after laser injury in the CNV model, lesion size was assessed by fundus infrared (IR) imaging (left) before Evans Blue was injected intraperitoneally and dye leakage visualized 24 h later in eye sections counterstained with IB4 and DAPI; the Evans Blue single channel is shown in grayscale on the right hand side. Leakage into the retina at lesion level (as indicated by red) was quantified as the number of Evans Blue–positive pixels integrated for Evans Blue pixel intensity in mutants relative to littermate controls; mean ± SEM; n ≥ 8 mice each; *, P < 0.05 (unpaired Student’s t test). (E) Maximum intensity projections of confocal z-stacks through whole mount RPE/choroids from Nrp1 cyto/cyto and wild-type littermates stained for IB4 on D14 after lasering in the CNV model. Quantification of lesion size (right) as number of IB4-positive pixels integrated for IB4-pixel intensity in mutants relative to littermate controls; mean ± SEM; n ≥ 5 eyes each; ns, not significant; P > 0.05 (unpaired Student’s t test). Bars: 25 µm (A); 1 mm (D, left); 200 µm (D, right); 200 µm (E).

Article Snippet: HDMEC and MLEC were fixed in 4% formaldehyde in PBS for 15 min. To detect NRP1 in HDMEC, we used mouse anti–human NRP1 (R&D Systems).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Imaging, Injection

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Neuropilin 1 expression correlates with differentiation status of epidermal cells and cutaneous squamous cell carcinomas.

doi: 10.1038/labinvest.2014.66

Figure Lengend Snippet: Figure 2 Neuropilin 1 (NRP1) expression in human squamous cell carcinoma (SCC) patient samples correlates with degree of differentiation. (a) Human skin cancer tissue microarray A216 was stained by immunohistochemistry (IHC) for NRP1 protein expression. Illustrated is the scan of the entire slide showing 102 sections/biopsies. Six samples are marked with a line on the left and are shown at higher magnification below. (b) Scan of the entire section of highly differentiated (HD) human SCC sample stained by IHC for NRP1. Tumor shows high expression of NRP1 (brown color). (c) Selected samples from microarray in a demonstrate high NRP1 expression in normal (NR) epidermis and HD SCC samples, medium NRP1 expression in moderately differentiated (MD) samples, and the lack of NRP1 expression in poorly differentiated (PD) samples. Scale bar ¼ 200 mm.

Article Snippet: Membranes were blocked with non-fat milk and incubated with rabbit polyclonal anti-human NRP1 (44-2) (recognizing amino acids DDSKRKAKSFEGNNNYD in the b2 domain; not commercial)18 or goat anti-rat/mouse Nrp1 (R&D Systems).

Techniques: Expressing, Microarray, Staining, Immunohistochemistry

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Neuropilin 1 expression correlates with differentiation status of epidermal cells and cutaneous squamous cell carcinomas.

doi: 10.1038/labinvest.2014.66

Figure Lengend Snippet: Figure 3 Differentiated cells of human squamous cell carcinoma (SCC) xenografts in mice express the highest levels of neuropilin 1 (NRP1). Three human SCC cell lines were grown in nude mice: well-differentiated SCC13 (a, d, and g), moderately differentiated DJM1 (b, e, and h), and poorly differentiated A431 (c, f, and i). Paraffin sections of tumors were stained by immunohistochemistry (IHC) for human NRP1 (a–c), human keratin 1 (K1) (d–f), and human K14 (g–i). Note that human NRP1 antibodies do not stain mouse blood vessels. NRP1 is absent from poorly differentiated human SCC cells (arrows in a and d). Scale bar ¼ 100 mm.

Article Snippet: Membranes were blocked with non-fat milk and incubated with rabbit polyclonal anti-human NRP1 (44-2) (recognizing amino acids DDSKRKAKSFEGNNNYD in the b2 domain; not commercial)18 or goat anti-rat/mouse Nrp1 (R&D Systems).

Techniques: Staining, Immunohistochemistry

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Neuropilin 1 expression correlates with differentiation status of epidermal cells and cutaneous squamous cell carcinomas.

doi: 10.1038/labinvest.2014.66

Figure Lengend Snippet: Figure 4 Nrp1 is expressed in normal mouse epithelium and murine squamous cell carcinoma (SCC). Transgenic K14-HPV16 mice develop spontaneous and progressive dysplasia and SCC. Paraffin sections of the ears from K14-HPV16 mice were obtained before overt disease (normal) (a, d, and g), from dysplastic ears (b, e, and h), or from SCC tumors (c, f, and i). Sections were stained with hematoxylin and eosin (H&E) (a–c) or immunostained for mouse Nrp1 (brown color) (d–f), or mouse CD31 (brown color) (g–i). Nrp1 expression increased in dysplastic lesions (e) and was expressed in differentiated areas of SCC (f). Microvessel density progressively increased from normal/hyperplastic epidermis (g) to dysplasia (h), to SCC (i). Some sections (d–i) were counterstained with hematoxylin (blue color). All images were taken at 200 magnification; scale bars ¼ 100 mm. Notice the ears increase dramatically in thickness.

Article Snippet: Membranes were blocked with non-fat milk and incubated with rabbit polyclonal anti-human NRP1 (44-2) (recognizing amino acids DDSKRKAKSFEGNNNYD in the b2 domain; not commercial)18 or goat anti-rat/mouse Nrp1 (R&D Systems).

Techniques: Transgenic Assay, Staining, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Neuropilin 1 expression correlates with differentiation status of epidermal cells and cutaneous squamous cell carcinomas.

doi: 10.1038/labinvest.2014.66

Figure Lengend Snippet: Figure 6 Nrp1 mRNA expression increased following UVB irradiation. Paraffin sections of mouse ears from normal, non-irradiated (control) (a and d), or UVB irradiated mice (b, c, e and f). Sections were stained with hematoxylin and eosin (H&E) (a–c) or by ISH with probes to mouse Nrp1 mRNA (purple color). Nrp1 expression was increased in epidermal cells and dermal vessels after UVB irradiation (e and f). Nrp1 mRNA expression was seen only in suprabasal cells of the epidermis. There was low Nrp1 expression in normal ears at short chromogen incubations (d) but the expression was detectable when incubation time was increased (not shown). All images were taken at 200 magnification; scale bars ¼ 100 mm. Notice the ears increase dramatically in thickness.

Article Snippet: Membranes were blocked with non-fat milk and incubated with rabbit polyclonal anti-human NRP1 (44-2) (recognizing amino acids DDSKRKAKSFEGNNNYD in the b2 domain; not commercial)18 or goat anti-rat/mouse Nrp1 (R&D Systems).

Techniques: Expressing, Irradiation, Control, Staining, Incubation

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Neuropilin 1 expression correlates with differentiation status of epidermal cells and cutaneous squamous cell carcinomas.

doi: 10.1038/labinvest.2014.66

Figure Lengend Snippet: Figure 7 Neuropilin 1 (NRP1) expression is regulated by differentiation and growth factors in vitro. (a–c) Primary mouse keratinocytes were isolated and cultured from P3 Balb/c mice. Cells were grown in (a) low-calcium growth media or differentiating high-calcium media for (b) 1 to (c) 3 days. Phase-contrast microscopy demonstrated changes in cell shape and morphology. (d) Western blotting of protein lysates from primary mouse keratinocytes cultured in low- (lo ¼ 0.05 mM) or high- (hi ¼ 0.12 mM) calcium media. Nrp1 protein is upregulated in cells grown in the differentiating media. (e and f) Northern blotting of mRNA from primary mouse keratinocytes cultured in (e) high-calcium media or (f) retinoic acid (RA) for various time points. Nrp1 was induced in cells grown for 1 day in high-calcium media or 4 days in RA. (g and h) Northern blotting of mRNA from primary mouse keratinocytes (g) or human Hacat cells (h) incubated for various times in growth factor. Addition of HB-EGF or epidermal growth factor (EGF) upregulated NRP1 expression in keratinocytes. (i) I125-VEGF165 cross-linking to NRP1 on Hacat cells increased after pretreatment in HB-EGF (left panel). I125-VEGF165 cross-linking to NRP1 on Hacat cells was competed in the presence of excess SEMA3A protein (right panel). Arrowheads point to the cross- linked complex of NRP1/VEGF.

Article Snippet: Membranes were blocked with non-fat milk and incubated with rabbit polyclonal anti-human NRP1 (44-2) (recognizing amino acids DDSKRKAKSFEGNNNYD in the b2 domain; not commercial)18 or goat anti-rat/mouse Nrp1 (R&D Systems).

Techniques: Expressing, In Vitro, Isolation, Cell Culture, Microscopy, Western Blot, Northern Blot, Incubation