Journal: Cell Death & Disease

Article Title: In vivo self-assembly and delivery of VEGFR2 siRNA-encapsulated small extracellular vesicles for lung metastatic osteosarcoma therapy

doi: 10.1038/s41419-023-06159-3

Figure Lengend Snippet: A Schematic of the experimental design. C57BL/6 J mice were intravenously injected with scrRNA circuit or anti-VEGFR2 circuit (10 mg/kg) every 2 days for a total of seven times, and then the sEVs were purified from mouse serum and incubated with HUVECs. Next, the uptake of self-assembled VEGFR2 siRNA by HUVECs and the subsequent suppression of VEGFR2 expression by self-assembled VEGFR2 siRNA were examined in this ex vivo model. B Representative TEM images of serum sEVs. Scale bar: 100 nm. C The size distribution and concentration of serum sEVs were determined by NTA. D Western blot analysis of specific markers (Alix, TSG101 and CD9) in purified sEVs and sEV-depleted serum. E qRT-PCR analysis of VEGFR2 siRNA levels in serum sEVs ( n = 3 per group). F , G Serum sEV RNA in C57BL/6 J mice injected with an anti-VEGFR2 circuit was subjected to immunoprecipitation using IgG or anti-AGO2 beads. Representative western blots ( F ) and qRT-PCR analysis data ( G ) are shown ( n = 3 per group). H Serum sEVs were fluorescently labeled with PKH26, and PKH26-labeled sEVs were incubated with HUVECs for 6 h. The level of intracellular fluorescence intensity was monitored by confocal microscopy. Scale bar: 50 μm. I , J Western blot analysis of VEGFR2 protein levels in HUVECs after 36- h of incubation with serum sEVs. Different doses of sEVs were added to reveal the dose-dependent effect. Representative western blots ( I ) and densitometric analysis data ( J ) are shown ( n = 3 per group). K qRT–PCR analysis of VEGFR2 mRNA levels in HUVECs after 36- h of incubation with different doses of serum sEVs ( n = 3 per group). Values are presented as the mean ± SD. Significance was determined using a two-sided t -test in G or using one-way ANOVA in J and K . * P < 0.05; ** P < 0.01; **** P < 0.0001; ns = not significant.

Article Snippet: The human umbilical vein endothelial cell line HUVEC (RRID: CVCL_2959, female), mouse endothelial cell line EOMA (RRID: CVCL_3507), and human osteosarcoma cell line 143B (RRID: CVCL_2270, female) were purchased from American Type Culture Collection (ATCC).

Techniques: Injection, Purification, Incubation, Expressing, Ex Vivo, Concentration Assay, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Labeling, Fluorescence, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: In vivo self-assembly and delivery of VEGFR2 siRNA-encapsulated small extracellular vesicles for lung metastatic osteosarcoma therapy

doi: 10.1038/s41419-023-06159-3

Figure Lengend Snippet: A Schematic of the experimental design. Serum sEVs were harvested from C57BL/6 J mice injected with scrRNA circuit or anti-VEGFR2 circuit (10 mg/kg) for a total of seven times, and were subsequently subjected to evaluation of the intrinsic antiangiogenic capacity by incubating with HUVECs, mouse aortic rings and chicken allantoic membrane (CAM). B A CCK-8 assay was performed to estimate cell viability after incubation with serum sEVs from C57BL/6 J mice injected with scrRNA circuit or anti-VEGFR2 circuit at different time points in HUVECs ( n = 6 per time point). C , D A EdU assay was applied to estimate cell proliferation after incubation with serum sEVs in HUVECs. S-phase entry is visualized by EdU incorporation (green); DAPI-stained nuclei (blue). Scale bar: 200 μm. Quantification of EdU incorporation is shown in D ( n = 3 per group). E – G Wound healing and Transwell migration assays exhibited cell migration capability after incubation with serum sEVs in HUVECs. Scale bars: 200 μm ( E ) and 400 μm ( F ). Quantification of Transwell migration is shown in G ( n = 3 per group). H , I Tube formation assay showed cell tube formation ability after incubation with serum sEVs in HUVECs. Scale bar: 1 mm. Image quantification was conducted as described in methods ( I ) ( n = 3 per group). J , K Mouse aortic ring assay revealed the angiogenic ability of mouse aortic rings after incubation with serum sEVs. Representative immunofluorescence images ( J ). BS1 lectin-FITC (green) indicates endothelial sprouts; α-SMA represents supporting cells (red); DAPI-stained nuclei (blue). Scale bar: 400 μm. Quantification of aortic ring microvessel area as described in methods ( K ) ( n = 5 per group). L , M The CAM assay revealed the angiogenic ability of CAM after incubation with serum sEVs. Representative images of CAM photographed on plastic dishes after resection from eggs ( L ). Scale bar: 2 cm. The statistical results of the CAM assay ( M ) ( n = 5 per group). Values are presented as the mean ± SD. Significance was determined using a two-sided t -test in B , D , G and K or using one-way ANOVA in I and M . * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns = not significant.

Article Snippet: The human umbilical vein endothelial cell line HUVEC (RRID: CVCL_2959, female), mouse endothelial cell line EOMA (RRID: CVCL_3507), and human osteosarcoma cell line 143B (RRID: CVCL_2270, female) were purchased from American Type Culture Collection (ATCC).

Techniques: Injection, Membrane, CCK-8 Assay, Incubation, EdU Assay, Staining, Migration, Tube Formation Assay, Aortic Ring Assay, Immunofluorescence, Chick Chorioallantoic Membrane Assay

Journal: Cell Research

Article Title: CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development

doi: 10.1038/cr.2015.15

Figure Lengend Snippet: Netrin-1 binds to CD146. (A) Co-immunoprecipitation assays. Netrin-1- and CD146-expressing plasmids were co-transfected into HEK293 cells prior to preparation of cell lysates. Lanes 1 and 4: precipitated by control mIgG. Lanes 2 and 3: anti-CD146 mAb AA1. Lanes 5 and 6: anti-netrin-1 mAb. (B) HEK293 cells were transfected with plasmids encoding DCC, CD146 or Robo-1 and incubated with netrin-1-GFP conditional medium. Binding of netrin-1-GFP to the cell was detected by its GFP fluorescence. Scale bar, 50 μm. (C) Direct interaction between purified netrin-1 and CD146 proteins in vitro . Fc, Fc-CD146 or Fc-UNC5B (200 ng/ml) bound protein G beads was incubated with netrin-1 protein (200 ng/ml). The bound proteins were analyzed by western blotting. (D , E) Determination of netrin-CD146 binding affinity by SPR. Purified netrin-1 protein was applied at different concentrations to the CM5 chips containing Fc-CD146 (D) or Fc-UNC5B (E) . Data represent 3 independent experiments.

Article Snippet: The following reagents were used: recombinant hnetrin-1, mouse netrin-1 and Fc-UNC5B (Enzo Life Science); human Fc, Fc-CD146, sCD146 (Sino Biological), growth factor-reduced Matrigel (BD Biosciences); Fugene HD, DAPI and protease inhibitor cocktails (Roche); protein G sepharose beads and DAB substrates (Santa Cruz); Enhanced Chemiluminescence Assay Kit (Pierce) for western blotting; cell counting kit-8 (CCK8, Dojindo) for cell proliferation assay.

Techniques: Immunoprecipitation, Expressing, Transfection, Incubation, Binding Assay, Fluorescence, Purification, In Vitro, Western Blot

Journal: Cell Research

Article Title: CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development

doi: 10.1038/cr.2015.15

Figure Lengend Snippet: Identification of interacting domains of netrin-1 and CD146. (A) A diagrammatic illustration of the full-length (FL) and truncation mutants of netrin-1 expressed as His-tagged proteins. (B) HEK293 cells were co-transfected with plasmid expressing CD146 and the FL or truncation mutant of netrin-1. The cell lysates were prepared for immunoprecipitation with anti-CD146 mAb AA1. (C , D) Defining the domain of CD146 required for interaction with netrin-1. CD146 and its truncation mutants were expressed as Flag-tagged proteins. Immunoprecipitation was performed using anti-netrin-1 mAb. Data represent 3 independent experiments.

Article Snippet: The following reagents were used: recombinant hnetrin-1, mouse netrin-1 and Fc-UNC5B (Enzo Life Science); human Fc, Fc-CD146, sCD146 (Sino Biological), growth factor-reduced Matrigel (BD Biosciences); Fugene HD, DAPI and protease inhibitor cocktails (Roche); protein G sepharose beads and DAB substrates (Santa Cruz); Enhanced Chemiluminescence Assay Kit (Pierce) for western blotting; cell counting kit-8 (CCK8, Dojindo) for cell proliferation assay.

Techniques: Transfection, Plasmid Preparation, Expressing, Mutagenesis, Immunoprecipitation

Journal: Cell Research

Article Title: CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development

doi: 10.1038/cr.2015.15

Figure Lengend Snippet: Netrin-1 exhibits dual activities in endothelial cell activation. (A-C) HUVECs were used for proliferation assay (A) , transwell migration assay (B) and tube formation assay (C) . Netrin-1 was applied at concentrations as indicated. (D) serum-starved HUVECs were treated with netrin-1 at different concentrations for 10 min. Dimerization of CD146 and phosphorylation of VEGFR2, ERK1/2 and p38 were analyzed by western blotting. The black arrow marks the CD146 dimer. (E) Representative images of netrin-1-induced HUVEC migration (left) and tube formation (right). For A-C , n = 3 in each group. Data represent 3 independent experiments (means ± SEM). * P < 0.05; ** P < 0.01; *** P < 0.001 (one-way ANOVA with Turkey post hoc tests).

Article Snippet: The following reagents were used: recombinant hnetrin-1, mouse netrin-1 and Fc-UNC5B (Enzo Life Science); human Fc, Fc-CD146, sCD146 (Sino Biological), growth factor-reduced Matrigel (BD Biosciences); Fugene HD, DAPI and protease inhibitor cocktails (Roche); protein G sepharose beads and DAB substrates (Santa Cruz); Enhanced Chemiluminescence Assay Kit (Pierce) for western blotting; cell counting kit-8 (CCK8, Dojindo) for cell proliferation assay.

Techniques: Activation Assay, Proliferation Assay, Transwell Migration Assay, Tube Formation Assay, Western Blot, Migration

Journal: Cell Research

Article Title: CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development

doi: 10.1038/cr.2015.15

Figure Lengend Snippet: CD146 mediates netrin-1-induced endothelial cell activation. (A – D) HUVECs transfected with control, CD146, UNC5B or VEGFR2 specific siRNA were subjected to proliferation assay (A) , transwell migration assay (B) , tube formation assay (C) and signaling activation assay (D) . Netrin-1 was applied at the indicated concentrations. Note that the specific siRNAs efficiently downregulated the expression of corresponding molecules. For A – C , n = 3 in each group. Data represent 3 independent experiments (means ± SEM). * P < 0.05; ** P < 0.01; *** P < 0.001; 'NS' indicates no significant changes observed (one-way ANOVA with Turkey post hoc tests).

Article Snippet: The following reagents were used: recombinant hnetrin-1, mouse netrin-1 and Fc-UNC5B (Enzo Life Science); human Fc, Fc-CD146, sCD146 (Sino Biological), growth factor-reduced Matrigel (BD Biosciences); Fugene HD, DAPI and protease inhibitor cocktails (Roche); protein G sepharose beads and DAB substrates (Santa Cruz); Enhanced Chemiluminescence Assay Kit (Pierce) for western blotting; cell counting kit-8 (CCK8, Dojindo) for cell proliferation assay.

Techniques: Activation Assay, Transfection, Proliferation Assay, Transwell Migration Assay, Tube Formation Assay, Expressing

Journal: Cell Research

Article Title: CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development

doi: 10.1038/cr.2015.15

Figure Lengend Snippet: CD146 is required for netrin-1-induced angiogenesis in mouse models. (A) Aortic rings were prepared from WT or CD146 EC-KO mice. Control or netrin-1 (50 ng/ml) was directly added to the culture medium. (B) The effect of anti-CD146 antibody AA98 was tested in the aortic-ring assay. Control mIgG or AA98 (100 μg/ml) was added to the culture medium in the presence of control or netrin-1 (50 ng/ml). After culturing for 5-6 days, the number of sprouts from each ring was quantified. n = 10 in each group and results are presented as average number of sprouts per ring (means ± SEM). (C) The Matrigel-plug assay for angiogenesis was carried out using WT or CD146 EC-KO mice. The plugs were mixed with control or netrin-1 (200 ng/ml) and then injected subcutaneously into mice in the corresponding groups. (D) The effect of anti-CD146 antibody AA98 on netrin-1-induced angiogenesis was tested in the Matrigel-plug assay. The plugs were pre-mixed with AA98 or control mIgG (100 μg/ml) and injected into the WT mice. 10 days post injection, the Matrigel plugs were sectioned and immunostained with anti-CD31 antibody. The number of blood vessels in each section was scored. n = 5 in each group and results are presented as average number of blood vessels/mm 2 (means ± SEM). Scale bar, 200 μm. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: The following reagents were used: recombinant hnetrin-1, mouse netrin-1 and Fc-UNC5B (Enzo Life Science); human Fc, Fc-CD146, sCD146 (Sino Biological), growth factor-reduced Matrigel (BD Biosciences); Fugene HD, DAPI and protease inhibitor cocktails (Roche); protein G sepharose beads and DAB substrates (Santa Cruz); Enhanced Chemiluminescence Assay Kit (Pierce) for western blotting; cell counting kit-8 (CCK8, Dojindo) for cell proliferation assay.

Techniques: Aortic Ring Assay, Matrigel Assay, Injection

Journal: Cell Research

Article Title: CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development

doi: 10.1038/cr.2015.15

Figure Lengend Snippet: CD146 mediates netrin-1-induced angiogenesis in zebrafish. (A) CD146 expression in the zebrafish embryos at different stages was detected by whole mount in situ hybridization. (B) The control, CD146 or netrin-1a specific MO was injected with control or human netrin-1 mRNA into the transgenic zebrafish Tg(kdrl:GFP) embryos that expressed GFP to enable visualization of vasculature. The vascular patterns of the fish embryos at 36, 48 and 60 hpf were analyzed. Yellow arrowheads indicate PAV. Red arrowheads mark ectopic sprouts in netrin-1 mRNA-injected embryos. (C) Tg(Fli1:nGFP) × Tg(kdrl:mcherry) embryos were injected with the indicated MO and mRNA. The number of endothelial cells in the ISVs of the embryos at 36 hpf, 48 hpf and 60 hpf stages was quantified and analyzed. Data are presented as average number of endothelial cells per ISV (means ± SEM). * P < 0.05; ** P < 0.01; *** P < 0.001 (one-way ANOVA with Turkey post hoc tests).

Article Snippet: The following reagents were used: recombinant hnetrin-1, mouse netrin-1 and Fc-UNC5B (Enzo Life Science); human Fc, Fc-CD146, sCD146 (Sino Biological), growth factor-reduced Matrigel (BD Biosciences); Fugene HD, DAPI and protease inhibitor cocktails (Roche); protein G sepharose beads and DAB substrates (Santa Cruz); Enhanced Chemiluminescence Assay Kit (Pierce) for western blotting; cell counting kit-8 (CCK8, Dojindo) for cell proliferation assay.

Techniques: Expressing, In Situ Hybridization, Injection, Transgenic Assay

Journal: Physiological Reports

Article Title: Chemerin promotes angiogenesis in vivo

doi: 10.14814/phy2.13962

Figure Lengend Snippet: Chemerin promotes angiogenesis in vivo. (A) Matrigel plugs containing chemerin (10 nmol/L, n = 6), VEGF (5 nmol/L, n = 6), or phosphate‐buffered saline (control, n = 6) were injected subcutaneously into mice. Plugs were stained with the endothelial cell marker CD 31 and vWF (arrow indicated). Bar = 100 μ m. (B) Endothelial cell infiltration of the plugs was assessed by immunohistochemical analysis of CD 31 and vWF positive cells. The frequency of DAPI (+) CD 31 (+) vWF (+) cells in five low power fields was determined for each Matrigel plug. The data were presented as the number of DAPI (+) CD 31 (+) vWF (+) cells per microscopic area. The mean ± SD was derived from six Matrigel plugs for each condition. * P < 0.05 compared with control. (C) Mouse corneal angiogenesis assay; Pellets containing chemerin (200 ng, n = 6), VEGF (100 ng, n = 6) were implanted in mouse cornea. Photographs of mouse eyes are shown (Control, chemerin 200 ng; VEGF , 100 ng). (D) The angiogenic activity was evaluated based on the number of newly formed capillaries per cornea. The results are shown as the mean ± SE ** P < 0.01 versus control. (D) Rat aortic ring assay; The descending thoracic aorta of single male Sprague–Dawley rat was removed, embedded in Matrigel, and incubated with M199 medium containing chemerin (10 nmol/L, n = 6), VEGF (5 nmol/L, n = 6). (E) Outgrowth of neovessels from the aorta was observed under phase‐contrast microscopy. (F) The length of neovessels from the aorta was calculated by the use of a cell image analyzer. ** P < 0.01 compared with control.

Article Snippet: Chemicals – Recombinant human chemerin (cat. No.: 2324‐CM), recombinant mouse chemerin (cat. No.: 2325‐CM), and VEGF‐A165 (cat.No:293‐VE) as positive controls were obtained from R&D systems (Abingdon, UK).

Techniques: In Vivo, Saline, Control, Injection, Staining, Marker, Immunohistochemical staining, Derivative Assay, Corneal Angiogenesis Assay, Activity Assay, Aortic Ring Assay, Incubation, Microscopy

Journal: Physiological Reports

Article Title: Chemerin promotes angiogenesis in vivo

doi: 10.14814/phy2.13962

Figure Lengend Snippet: (A) Expression of chemerin receptors, CMKLR 1 and CCRL 2, in HUVEC s. Chemerin receptors were identified by Western blot analyses using anti‐human CMKLR 1 and CCRL 2 antibodies. (B) The phosphorylation of Akt and ERK by chemerin. Chemerin stimulation (10 nmol/L) was conducted for the indicated time. The results of one of three experiments with similar results are shown. * P < 0.01 versus control. (C) Confocal immunofluorescence analysis of HUVEC s treated with chemerin or VEGF using rabbit mA b against phospho‐Akt (Ser473) and phospho‐ ERK (Thr202/Tyr204) (green). Actin filaments have been labeled with Alexa Fluor 568‐conjugated phalloidin (red). Blue pseudo color = DAPI.

Article Snippet: Chemicals – Recombinant human chemerin (cat. No.: 2324‐CM), recombinant mouse chemerin (cat. No.: 2325‐CM), and VEGF‐A165 (cat.No:293‐VE) as positive controls were obtained from R&D systems (Abingdon, UK).

Techniques: Expressing, Western Blot, Phospho-proteomics, Control, Immunofluorescence, Labeling

Journal: Physiological Reports

Article Title: Chemerin promotes angiogenesis in vivo

doi: 10.14814/phy2.13962

Figure Lengend Snippet: Migration of HUVEC s induced by chemerin. (A) Modified Boyden chamber assay was performed with chemerin (10 nmol/L) or VEGF (5 nmol/L) as chemoattractant. Chemerin stimulation (10 nmol/L) was conducted for the indicated time. Seven random microscopic fields per each well were quantified. All assays were performed in triplicate. The results are shown as the mean ± SE . * P < 0.05, ** P < 0.01 versus control. (B) Representative images of the migrated HUVEC in the modified Boyden assay. Cells were stained with CD 31 and DAPI (arrow indicated). Bar = 100 μ m. (C) Chemerin‐induced enhancement of migration. After preincubation with LY 294002 (1 μ mol/L) or PD 98059 (5 μ mol/L), cells were incubated with chemerin (10 nmol/L). The migratory capacity was quantified by counting the migrated HUVEC s on the lower surface of the filter. Seven random microscopic fields per each well were quantified. All assays were performed in triplicate. The results are shown as the mean ± SE . * P < 0.01 versus control.

Article Snippet: Chemicals – Recombinant human chemerin (cat. No.: 2324‐CM), recombinant mouse chemerin (cat. No.: 2325‐CM), and VEGF‐A165 (cat.No:293‐VE) as positive controls were obtained from R&D systems (Abingdon, UK).

Techniques: Migration, Modification, Boyden Chamber Assay, Control, Boyden Assay, Staining, Incubation

Journal: Physiological Reports

Article Title: Chemerin promotes angiogenesis in vivo

doi: 10.14814/phy2.13962

Figure Lengend Snippet: Migration of HUVEC s stimulated by chemerin assessed by cell scratch assay. (A) Modified wound healing assay was performed with chemerin (10 nmol/L) or VEGF (5 nmol/L) as chemoattractant. Chemerin stimulation (10 nmol/L) was conducted for the indicated time. After preincubation with LY 294002 (1 μ mol/L) or PD 98059 (5 μ mol/L), cells were incubated with chemerin (10 nmol/L). (B) The change rate of width between the leading edge covered by cells before and after 8 h of incubation was quantified. Seven random wounds per each well were quantified. All assays were performed in triplicate. * P < 0.05 versus serum‐free control.

Article Snippet: Chemicals – Recombinant human chemerin (cat. No.: 2324‐CM), recombinant mouse chemerin (cat. No.: 2325‐CM), and VEGF‐A165 (cat.No:293‐VE) as positive controls were obtained from R&D systems (Abingdon, UK).

Techniques: Migration, Wound Healing Assay, Modification, Incubation, Control

Journal: Physiological Reports

Article Title: Chemerin promotes angiogenesis in vivo

doi: 10.14814/phy2.13962

Figure Lengend Snippet: Chemerin promotes proliferation of HUVEC s. Cell growth was arrested using serum‐free EBM ‐2 medium for 12 h. After preincubation with LY 294002 (1 μ mol/L) or PD 98059 (5 μ mol/L), cells were incubated with chemerin (10 nmol/L) or VEGF (5 nmol/L). (A) Cell proliferation was assessed by counting cells with a hemocytometer. For cell counting, HUVEC s were detached from the each seven plates and counted using a hemocytometer. All assays were performed in triplicate. (B) Cell proliferation was assessed by the MTT assay. MTT reduction was read by each 10 wells at 550 nm using a spectrophotometer. All assays were performed in triplicate. The results are shown as the mean ± SE . * P < 0.05 versus control.

Article Snippet: Chemicals – Recombinant human chemerin (cat. No.: 2324‐CM), recombinant mouse chemerin (cat. No.: 2325‐CM), and VEGF‐A165 (cat.No:293‐VE) as positive controls were obtained from R&D systems (Abingdon, UK).

Techniques: Incubation, Cell Counting, MTT Assay, Spectrophotometry, Control

Journal: Physiological Reports

Article Title: Chemerin promotes angiogenesis in vivo

doi: 10.14814/phy2.13962

Figure Lengend Snippet: Chemerin promotes the differentiation of HUVEC s into tube‐like structures. Tube formation assays were performed. HUVEC s were seeded on Matrigel‐coated culture dishes in the presence of chemerin (10 nmol/L), VEGF (5 nmol/L), or BSA (10 g/mL: control). After preincubation with LY 294002 (1 μ mol/L) or PD 98059 (5 μ mol/L), cells were incubated with chemerin (10 nmol/L). (A) Representative cultures are shown. (B) Quantitative analysis of tube formation. Total tube length per each 10 microscopic area was calculated using image analyzer software. All assays were performed in triplicate. The results are shown as the mean ± SE . * P < 0.05 versus control.

Article Snippet: Chemicals – Recombinant human chemerin (cat. No.: 2324‐CM), recombinant mouse chemerin (cat. No.: 2325‐CM), and VEGF‐A165 (cat.No:293‐VE) as positive controls were obtained from R&D systems (Abingdon, UK).

Techniques: Control, Incubation, Software

Journal: Physiological Reports

Article Title: Chemerin promotes angiogenesis in vivo

doi: 10.14814/phy2.13962

Figure Lengend Snippet: (A) The si RNA ‐mediated knockdown of CMKLR 1 and CCRL 2. (B) Chemerin‐induced phosphorylation of Akt and ERK was inhibited by si RNA against CMKLR 1. (C) Modified Boyden chamber assay was performed with chemerin (10 nmol/L) as chemoattractant. Chemerin‐induced enhancement of migration was inhibited by si RNA against CMKLR 1. Seven random microscopic fields per each well were quantified. All assays were performed in triplicate. The results are shown as the mean ± SE . * P < 0.05 versus control.

Article Snippet: Chemicals – Recombinant human chemerin (cat. No.: 2324‐CM), recombinant mouse chemerin (cat. No.: 2325‐CM), and VEGF‐A165 (cat.No:293‐VE) as positive controls were obtained from R&D systems (Abingdon, UK).

Techniques: Knockdown, Phospho-proteomics, Modification, Boyden Chamber Assay, Migration, Control

Journal: Physiological Reports

Article Title: Chemerin promotes angiogenesis in vivo

doi: 10.14814/phy2.13962

Figure Lengend Snippet: Chemerin promotes the differentiation of HUVEC s into tube‐like structures via CMKLR 1. HUVEC s were seeded on Matrigel‐coated culture dishes in the presence of chemerin (10 nmol/L) Chemerin‐induced enhancement of angiogenesis was inhibited by si RNA against CMKLR 1. (A) Representative cultures are shown. (B) Quantitative analysis of tube formation. Total tube length per each 10 microscopic area was calculated using image analyzer software. All assays were performed in triplicate. The results are shown as the mean ± SE . * P < 0.05 versus control.

Article Snippet: Chemicals – Recombinant human chemerin (cat. No.: 2324‐CM), recombinant mouse chemerin (cat. No.: 2325‐CM), and VEGF‐A165 (cat.No:293‐VE) as positive controls were obtained from R&D systems (Abingdon, UK).

Techniques: Software, Control

Journal: Physiological Reports

Article Title: Chemerin promotes angiogenesis in vivo

doi: 10.14814/phy2.13962

Figure Lengend Snippet: Proposed scheme for chemerin‐stimulated signaling of proliferation and migration. Chemerin promotes angiogenesis and proliferation activity of HUVEC s via MEK / ERK and PI 3‐K/Akt, whereas chemerin promotes the migration activity only via PI 3‐K/Akt.

Article Snippet: Chemicals – Recombinant human chemerin (cat. No.: 2324‐CM), recombinant mouse chemerin (cat. No.: 2325‐CM), and VEGF‐A165 (cat.No:293‐VE) as positive controls were obtained from R&D systems (Abingdon, UK).

Techniques: Migration, Activity Assay

Journal: The Journal of Cell Biology

Article Title: VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia

doi: 10.1083/jcb.200302047

Figure Lengend Snippet: Illustration of tip cell guidance toward VEGF sources and of VEGFR expression on endothelial filopodia. (a–c) Confocal laser scanning micrographs of VEGF-A in situ hybridization (black signal) combined with double labeling for isolectin and GFAP. (a) Overview illustrating strong VEGF-A expression ahead of the vascular plexus and very low behind the leading edge. (b) Higher magnification showing astrocytic VEGF-A expression (black and red overlap) and strong down-regulation in astrocytes covered by the primary plexus. (c) Tip cell filopodia orientate toward and along VEGF-A–expressing astrocytes. (d) VEGFR2 in situ hybridization identifies strongest expression in the tip cells (arrows). (e and f) VEGFR2 is prominent on tip cell filopodia (arrowheads). (e) Rat mAb labeling using tyramide enhancer kit on cryosection, and (f) goat polyclonal antibody on whole mount labeling together with isolectin.

Article Snippet: The following substances were used: Flt-1/Fc chimera (471-F1–100; R&D Systems), neutralizing VEGFR1 antibody (AF471; R&D Systems), neutralizing VEGFR2 (AF644; R&D Systems), recombinant mouse VEGF-A 164 (493-MV/CF; R&D Systems), recombinant mouse PlGF-2 (465-PL/CF; R&D Systems), VEGF-E NZ2, VEGF-E NZ7, and VEGF-C156S (see Protein production).

Techniques: Expressing, In Situ Hybridization, Labeling

Journal: The Journal of Cell Biology

Article Title: VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia

doi: 10.1083/jcb.200302047

Figure Lengend Snippet: Illustration of filopodia induction in hyaloid vessels of VEGF164tg. (a) Wild-type littermate showing normal smooth surface of the hyaloid vessels (arrows) lying on the inner surface of the retina. Filopodia are only present in the intraretinal vascular plexus (asterisks). (b and c) Hyaloid vessels in VEGF164tg are studded with filopodia (arrows and inset). Bundles of filopodia are involved in sprouting and fusion (arrowhead), leading to an aberrant hyperfused vascular structure. Bars, 20 μm. VEGF is necessary for filopodia extension: (d–g) acute sequestering of VEGF by intraocular injection of soluble Flt-1–IgG chimeric protein leads to filopodia retraction already after 6 h (earliest time point investigated). (h–k) VEGF sequestering inhibits tip cell filopodia in the aortic ring assay. (l–o) Acute neutralization of VEGFR2 (n and o) but not VEGFR1 (l and m) leads to retraction of tip cell filopodia.

Article Snippet: The following substances were used: Flt-1/Fc chimera (471-F1–100; R&D Systems), neutralizing VEGFR1 antibody (AF471; R&D Systems), neutralizing VEGFR2 (AF644; R&D Systems), recombinant mouse VEGF-A 164 (493-MV/CF; R&D Systems), recombinant mouse PlGF-2 (465-PL/CF; R&D Systems), VEGF-E NZ2, VEGF-E NZ7, and VEGF-C156S (see Protein production).

Techniques: Injection, Aortic Ring Assay, Neutralization

Journal: The Journal of Cell Biology

Article Title: VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia

doi: 10.1083/jcb.200302047

Figure Lengend Snippet: Receptor specificity. (a) Binding of VEGF-E to VEGFR–IgG fusion proteins. VEGFR–IgG fusion proteins were incubated with purified histidine-tagged growth factors and precipitated with protein A sepharose. After reducing SDS-PAGE and Western blotting, receptor-bound growth factors were detected with pentahistidine antibodies. Both forms of VEGF-E (NZ2 and NZ7) show significant interaction only with VEGFR-2, whereas VEGF-A interacts additionally with VEGFR-1 and VEGF-C additionally with VEGFR-3. (b–i) Intraocular injection of VEGFR–specific ligands at 1 μg/μl concentrations. Retinas were fixed after 24 h. (b) Human serum albumin injection has no effect on patterning or filopodia (f). (c) VEGFR1-specific ligand leads to macrophage recruitment (round cells) but has no effect on patterning or tip cell filopodia (g). (d) VEGFR2-specific ligand mimics VEGF-A injection and VEGF120/120 retinas (compare with ). Note the lack of sprouting but increased vessel diameter and density. Only few very short filopodia are present (h, arrow). (e) VEGFR-3–specific ligand VEGF-C156S had no effect on patterning or tip cell filopodia (i). (j and k) Spreading of the vascular plexus over the retina is a measure of migration. Under all conditions of disturbed VEGF gradients, the spreading of the vasculature toward the periphery is significantly reduced. Examples are shown for VEGF-E injection. Overview pictures were taken on P6. Spreading distance as measured from the optic disc is indicated by red doubled arrow.

Article Snippet: The following substances were used: Flt-1/Fc chimera (471-F1–100; R&D Systems), neutralizing VEGFR1 antibody (AF471; R&D Systems), neutralizing VEGFR2 (AF644; R&D Systems), recombinant mouse VEGF-A 164 (493-MV/CF; R&D Systems), recombinant mouse PlGF-2 (465-PL/CF; R&D Systems), VEGF-E NZ2, VEGF-E NZ7, and VEGF-C156S (see Protein production).

Techniques: Binding Assay, Incubation, Purification, SDS Page, Western Blot, Injection, Migration

Journal: Stem cells (Dayton, Ohio)

Article Title: Heparanase Released From Mesenchymal Stem Cells Activates Integrin beta1/HIF-2alpha/Flk-1 Signaling and Promotes Endothelial Cell Migration and Angiogenesis

doi: 10.1002/stem.1995

Figure Lengend Snippet: MSC hpa promotes vascular regeneration in vivo. (A) : Representative Laser-Doppler images (LDI) of hind limbs before, immediately after, and 3, 7, and 14 days after femoral artery occlusion. (B) : Quantitative LDI analysis showing the right-to-left ( R/L ) ratio; n ≥ for each group, * denotes p < .05. (C) : Detection of heparanase protein expression using Western blot in WT MSC, MSC harboring empty vector, and heparanase over-expressed vector. (D) : Detection of heparanase by ELISA in conditioned medium of WT MSC, MSC harboring empty vector, and heparanase over-expressed vector; n = 4 for each group, * denotes p < .05. (E) : HE and immunofluorescent staining of α -SMA and CD31 in muscle tissues from each group; Bar = 100 μm for HE and Bar = 50 μm for immunofluorescent staining. (F, G) : Bar graph showed quantitative analysis of SMA and CD31 positive area density; n = 5 for each group, * denotes p < .05; ** denotes p < .01. Abbreviations: MSC, mesenchymal stem cell; PBS, phosphate buffer saline; SMA, smooth muscle actin.

Article Snippet: The lower chamber was filled with MSC-conditioned medium or control medium as above or with recombinant human active heparanase (100 mg/ml, R&D Systems, Minneapolis, MN, USA).

Techniques: In Vivo, Expressing, Western Blot, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Staining, Saline

Journal: Stem cells (Dayton, Ohio)

Article Title: Heparanase Released From Mesenchymal Stem Cells Activates Integrin beta1/HIF-2alpha/Flk-1 Signaling and Promotes Endothelial Cell Migration and Angiogenesis

doi: 10.1002/stem.1995

Figure Lengend Snippet: Proangiogenic role of MSC hpa in vitro. (A) : Protein expression of heparanase and actin by Western blot in MSC null and MSC hpa-KD . (B) : Detection of heparanase concentration using ELISA in conditioned medium of MSC WT , MSC null , and MSC hpa-KD ; n = 4 for each group, * denotes p < .05. (C) : Representative images showing tube formation of human umbilical vein endothelial cells after cocultured with conditioned medium derived from MSC WT , MSC null , MSC hpa , MSC hpa-KD , respectively; Bar = 50 μm. (D) : Quantification of tube length in each group; n = 3 for each group, ** denotes p < .01. (E) : Aortic ring assay showing sprouting and branching in each group; Bar = 50 μm. (F, G) : Bar graph showed quantitative analysis of sprout length and outgrowth area, respectively; n = 3 for each group, * denotes p < .05, ** denotes p < .01. Abbreviation: MSC, mesenchymal stem cell.

Article Snippet: The lower chamber was filled with MSC-conditioned medium or control medium as above or with recombinant human active heparanase (100 mg/ml, R&D Systems, Minneapolis, MN, USA).

Techniques: In Vitro, Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Aortic Ring Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Heparanase Released From Mesenchymal Stem Cells Activates Integrin beta1/HIF-2alpha/Flk-1 Signaling and Promotes Endothelial Cell Migration and Angiogenesis

doi: 10.1002/stem.1995

Figure Lengend Snippet: HIF-2 α activation by heparanase requires integrin β 1. (A) : Western blot and quantification of integrin β 1 and β 3 expressions in HUVECs treated with conditioned medium from MSC null , MSC hpa , and MSC hpa-KD . (B) : HIF-2 α and integrin β 1 expressions are decreased in integrin β 1 knockdown HUVECs. (C) : Representative images and bar graph of cell migration in HUVECs transfected with vector or integrin β 1 shRNA lentivirus; Bar = 50 μm, n = 3, ** denotes p < .01. (D) : Quantification of VEGF expression in MSC WT , MSC null , MSC hpa-KD , and MSC hpa . (E) : Quantification of VEGF, PDGF-BB, HGF, and TGF- β concentrations by ELISA in conditioned medium of MSC WT , MSC null , MSC hpa-KD , and MSC hpa ; n = 4 for each group. * denotes p < .05 (hpa vs. null) and ## denotes p < .01 (hpa-KD vs. null). (F) : Viability of MSCs in different concentrations of OGT2115 by CCK-8 assay. (G) : Bar graph shows quantitative analysis of cell migration, respectively; n = 3 for each group. * denotes p < .05 (hpa vs. null), ## denotes p < .01 (hpa1OGT2115 vs. hpa). (H) : Detection of heparanase concentration by ELISA in conditioned medium of MSC WT , MSC null , MSC hpa , and MSC hpa + OGT2115; n = 4 for each group, * denotes p < .05 (hpa vs. null), # denotes p < .05 (hpa+OGT2115 vs. hpa). (I) : Detection of VEGF, PDGF-BB, HGF, and TGF- β concentrations by ELISA in conditioned medium of MSC hpa and MSC hpa with OGT2115, n = 4 for each group. (J, K) : Bar graph shows quantitative analysis of cell migration and tube formation in each group; n = 3, ** denotes p < .01, * denotes p < .05. (L) : Quantification of integrin β 1, Flk-1, and HIF-2 α expression in HUVECs after incubated with active heparanase; n = 3. Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; HUVEC, human umbilical vein endothelial cell; MSC, mesenchymal stem cell.

Article Snippet: The lower chamber was filled with MSC-conditioned medium or control medium as above or with recombinant human active heparanase (100 mg/ml, R&D Systems, Minneapolis, MN, USA).

Techniques: Activation Assay, Western Blot, Knockdown, Migration, Transfection, Plasmid Preparation, shRNA, Expressing, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Concentration Assay, Incubation, Modification

Journal: Stem cells (Dayton, Ohio)

Article Title: Heparanase Released From Mesenchymal Stem Cells Activates Integrin beta1/HIF-2alpha/Flk-1 Signaling and Promotes Endothelial Cell Migration and Angiogenesis

doi: 10.1002/stem.1995

Figure Lengend Snippet: Schematic of cell migration and angiogenesis by MSC-secreted heparanase. Abbreviation: MSC, mesenchymal stem cell.

Article Snippet: The lower chamber was filled with MSC-conditioned medium or control medium as above or with recombinant human active heparanase (100 mg/ml, R&D Systems, Minneapolis, MN, USA).

Techniques: Migration

Journal: Advanced Science

Article Title: Group 2 Innate Lymphoid Cells Protect Mice from Abdominal Aortic Aneurysm Formation via IL5 and Eosinophils

doi: 10.1002/advs.202206958

Figure Lengend Snippet: ILC2 deficiency blocks IL5‐dependent EOS generation. FACS analysis of A) splenic CD11b + Siglec‐F + EOS, B) CD11b + Ly6G + neutrophils, C) CD11b + Ly6C hi and CD11b + Ly6C lo monocytes, D) CD11c + MHC‐II + DCs, and E) CD4 + CD8 − and CD4 − CD8 + T cells from Il7r Cre/+ and Rora fl/fl Il7r Cre/+ mice after peri‐aortic CaPO 4 ‐induced AAA. F) Blood IL5 levels in Il7r Cre and Rora fl/fl Il7r Cre/+ mice at 7 d after peri‐aortic sham or CaPO 4 injury‐induced AAA. G) FACS analysis of CD11b + Siglec‐F + EOS in bone‐marrow cells from Rora fl/fl Il7r Cre/+ mice after 12 d of culture with or without ILC2 lysates from WT and Il5 −/− mice, or 2 ng mL −1 of IL5 as a positive control. Data are mean±SEM. n = 3–16 per group. * p < 0.05, ** p < 0.01, *** p < 0.001, A–E) nonparametric Mann–Whitney U test, or F,G) one‐way ANOVA test.

Article Snippet: Mouse aortic SMCs were plated onto an 8‐well chamber slide and pretreated with or without EOS lysates from WT mice at 1 × 10 6 EOS mL −1 , ILC2 lysates from WT and Il5 −/− mice at 2 × 10 4 ILC2 mL −1 , and 10 ng mL −1 mouse IL5 (405‐ML, R&D Systems) for 48 h. Then SMCs were incubated with or without 100 × 10 −6 m PDTC (P8765, Sigma‐Aldrich) for 5 h to induce apoptosis.

Techniques: Positive Control, MANN-WHITNEY

Journal: Advanced Science

Article Title: Group 2 Innate Lymphoid Cells Protect Mice from Abdominal Aortic Aneurysm Formation via IL5 and Eosinophils

doi: 10.1002/advs.202206958

Figure Lengend Snippet: ILC2 and EOS interactions with SMCs. A) TUNEL staining detected PDTC‐induced aortic SMC apoptosis with or without lysates from WT EOS, WT ILC2, Il5 −/− and Il13 −/− ILC2, or recombinant IL5 and IL13 (10 ng mL −1 ). Scale: 50 µm. B) Immunoblot analysis detected pre‐caspase‐3 and cleaved caspase‐3 in PDTC‐induced aortic SMC apoptosis under different conditions as indicated. C) Aortic SMC proliferation under the treatments as indicated. D) Immunoblot analysis detected total AKT and p‐AKT in aortic SMCs treated with or without FBS and other conditions as indicated. E) Immunoblot analysis of TGF‐ β ‐induced p‐Smad2/3 signaling from aortic SMCs pretreated with or without EOS lysates from WT mice or ILC2 lysates from WT and Il5 −/− mice. Representative images in panels (A), (B), (D), and (E) are shown to the left. The extra band in the p‐Smad3 blot after TGF‐ β treatment remains unknown. Data are mean±SEM. n = 10 per group. * p < 0.05, ** p < 0.01, *** p < 0.001, one‐way ANOVA test for normal distributed samples (after Shapiro–Wilk test), followed by LSD correction.

Article Snippet: Mouse aortic SMCs were plated onto an 8‐well chamber slide and pretreated with or without EOS lysates from WT mice at 1 × 10 6 EOS mL −1 , ILC2 lysates from WT and Il5 −/− mice at 2 × 10 4 ILC2 mL −1 , and 10 ng mL −1 mouse IL5 (405‐ML, R&D Systems) for 48 h. Then SMCs were incubated with or without 100 × 10 −6 m PDTC (P8765, Sigma‐Aldrich) for 5 h to induce apoptosis.

Techniques: TUNEL Assay, Staining, Recombinant, Western Blot

Journal: Advanced Science

Article Title: Group 2 Innate Lymphoid Cells Protect Mice from Abdominal Aortic Aneurysm Formation via IL5 and Eosinophils

doi: 10.1002/advs.202206958

Figure Lengend Snippet: ILC2 and EOS interactions with ECs, DCs, and monocytes. A) Immunoblot detected ICAM‐1 and VCAM‐1 expression from aortic ECs pretreated with or without EOS lysates from WT mice or ILC2 lysates from WT or Il5 −/− mice. B/C) Mouse aortic ring assay representative images and quantification. bFGF was used as positive controls. Scale: 400 µm. D) FACS analysis of bone‐marrow‐derived DCs cultured with or without EOS lysates and ILC2 lysates from WT mice. FACS analysis of CD11b + Ly6C hi monocytes after bone‐marrow‐derived monocytes were treated with E) LPS and INF‐ γ , and F) CD11b + Ly6C lo monocytes after cells were treated with IL4, with and without EOS lysates and ILC2 lysates from WT mice. Representative images in panels (A) and (D) are shown to the left. Data are mean±SEM. n = 3–7 per group. * p < 0.05, ** p < 0.01, *** p < 0.001, one‐way ANOVA test.

Article Snippet: Mouse aortic SMCs were plated onto an 8‐well chamber slide and pretreated with or without EOS lysates from WT mice at 1 × 10 6 EOS mL −1 , ILC2 lysates from WT and Il5 −/− mice at 2 × 10 4 ILC2 mL −1 , and 10 ng mL −1 mouse IL5 (405‐ML, R&D Systems) for 48 h. Then SMCs were incubated with or without 100 × 10 −6 m PDTC (P8765, Sigma‐Aldrich) for 5 h to induce apoptosis.

Techniques: Western Blot, Expressing, Aortic Ring Assay, Derivative Assay, Cell Culture

Journal: Advanced Science

Article Title: Group 2 Innate Lymphoid Cells Protect Mice from Abdominal Aortic Aneurysm Formation via IL5 and Eosinophils

doi: 10.1002/advs.202206958

Figure Lengend Snippet: Reconstitution of ILC2 or EOS reduces AAA growth in ILC2‐deficient Rora fl/fl Il7r Cre/+ mice. A) Aortic diameter and length, and representative images of Rora fl/fl Il7r Cre/+ mice treated with or without WT EOS or ILC2 from WT and Il5 −/− mice. Power: 1.000. B) ILC2 sorting strategy from WT splenocytes. ILC2 were defined as CD45 + Lin − ICOS + CD127 + KLRG1 + cells and isolated from CD45.1 transgenic mice. Donor CD45.1 + ILC2 cells were used to monitor their accumulation in Rora fl/fl Il7r Cre/+ mouse AAA lesions by CD45.1 immunofluorescent staining. C) FACS demonstrated the purity of bone‐marrow‐derived CD11b + Siglec‐F + EOS after 12 d differentiation. Donor CD45.1 + EOS were also detected in Rora fl/fl Il7r Cre/+ mouse AAA lesions using CD45.1 immunofluorescent staining. D) FACS analysis of blood donor CD45.1 + ILC2 or EOS in Rora fl/fl Il7r Cre/+ mice at 7 d after reconstitution and peri‐aortic CaPO 4 injury. E/F) FACS analysis of donor CD45.1 + EOS in blood and CD45.1 + ILC2 in aorta from Rora fl/fl Il7r Cre/+ recipient mice at different days (1, 3, 5, 7) after donor cell adoptive transfer. FACS analysis of G) CD11b + Siglec‐F + EOS, H) CD11b + Ly6C hi and CD11b + Ly6C lo monocytes, I) CD11c + MHC‐II + DCs, and J) CD11b + Ly6G + neutrophils in AAA lesions from Rora fl/fl Il7r Cre/+ mice with or without receiving donor EOS or ILC2 from WT and Il5 −/− mice. Data are mean±SEM, n = 10–25 per group, * p < 0.05, ** p < 0.01, *** p < 0.001, one‐way ANOVA test for normal distributed samples (after Shapiro–Wilk test) followed by A,F–I) LSD correction or E) nonparametric Mann–Whitney U test.

Article Snippet: Mouse aortic SMCs were plated onto an 8‐well chamber slide and pretreated with or without EOS lysates from WT mice at 1 × 10 6 EOS mL −1 , ILC2 lysates from WT and Il5 −/− mice at 2 × 10 4 ILC2 mL −1 , and 10 ng mL −1 mouse IL5 (405‐ML, R&D Systems) for 48 h. Then SMCs were incubated with or without 100 × 10 −6 m PDTC (P8765, Sigma‐Aldrich) for 5 h to induce apoptosis.

Techniques: Isolation, Transgenic Assay, Staining, Derivative Assay, Adoptive Transfer Assay, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Amino acid transporter LAT1 in tumor-associated vascular endothelium promotes angiogenesis by regulating cell proliferation and VEGF-A-dependent mTORC1 activation

doi: 10.1186/s13046-020-01762-0

Figure Lengend Snippet: Effects of LAT1 inhibition in aortic ring assay and in Matrigel plug assay. a Whole-mount immunofluorescence of LAT1 and Claudin-5 in aortic rings. Dashed lines ; edge of aortic rings. Asterisks ; autofluorescence from aortic rings. b Aortic ring assay performed in the presence or the absence of 50 μM JPH203. Bar graph ; quantification of endothelial sprouts. c Immunofluorescence of LAT1 and CD31 in Matrigel plug frozen section. d Fluorescent images of isolated Matrigel plugs implanted with or without 50 μM JPH203. Bar graph ; quantification of FITC fluorescence. a , c LAT1 was stained with mLAT1 ( c ) antibody. Nuclei were stained with DAPI ( blue , in merged image). b , n = 12; d , n = 16 for non-treated control (−), n = 13 for JPH203

Article Snippet: For western blotting using total membrane fractions of oocytes, following antibodies were used for the detection of LAT1 and 4F2hc: anti-mLAT1(R), anti-4F2hc (sc-7094, Santa Cruz Biotechnology), peroxidase-conjugated goat anti-rabbit IgG (111–035-003, Jackson ImmunoResearch), and peroxidase-conjugated mouse anti-goat IgG (205–035-108, Jackson ImmunoResearch).

Techniques: Inhibition, Aortic Ring Assay, Matrigel Assay, Immunofluorescence, Isolation, Fluorescence, Staining, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Amino acid transporter LAT1 in tumor-associated vascular endothelium promotes angiogenesis by regulating cell proliferation and VEGF-A-dependent mTORC1 activation

doi: 10.1186/s13046-020-01762-0

Figure Lengend Snippet: Suppression of endothelial sprouting in aortic ring assay by genetic ablation of LAT1. a LAT1 mRNA expression in Matrigel-embedded aortic rings of Lat1 fl/fl / rtTA3 / TetO-Cre mice analyzed on the day of embedding (Day 0) and three days later (Day 3), with or without DOX-treatment. b Aortic ring assay using Lat1 fl/fl / rtTA3 / TetO-Cre mice and control littermates ( Lat1 fl/fl / rtTA3 and Lat1 fl/fl / TetO-Cre ). c LAT1 mRNA expression in aortic rings of Lat1 fl/fl /Tek-Cre mice and control littermates ( Lat1 fl/fl ) analyzed on Day 0 and Day 3. d Aortic ring assay using Lat1 fl/fl /Tek-Cre and Lat1 fl/fl mice. b , d Bar graphs ; quantification of endothelial sprouts. a , c , n = 4; b , n = 5; d , n = 8

Article Snippet: For western blotting using total membrane fractions of oocytes, following antibodies were used for the detection of LAT1 and 4F2hc: anti-mLAT1(R), anti-4F2hc (sc-7094, Santa Cruz Biotechnology), peroxidase-conjugated goat anti-rabbit IgG (111–035-003, Jackson ImmunoResearch), and peroxidase-conjugated mouse anti-goat IgG (205–035-108, Jackson ImmunoResearch).

Techniques: Aortic Ring Assay, Expressing, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Amino acid transporter LAT1 in tumor-associated vascular endothelium promotes angiogenesis by regulating cell proliferation and VEGF-A-dependent mTORC1 activation

doi: 10.1186/s13046-020-01762-0

Figure Lengend Snippet: Anti-angiogenic effects of LAT1 inhibition and endothelial-specific LAT1 depletion in in vivo tumor models. a Growth of MIA PaCa-2 xenograft tumors in athymic nude mice. White bar indicates the period of daily drug administration. Control, placebo treatment; JPH203, 25 mg/kg/day i.v. ( n = 5 each). b Tissue weight and c gross appearance of xenograft tumors collected on Day 21. d Immunofluorescence of CD34 in the tumor paraffin sections. e Quantification of the density of CD34-positive intratumoral blood vessels. f Immunohistochemistry of LAT1 and CD34 in the paraffin sections of orthotopic syngeneic tumor model of B16-F10 cells in C57BL/6 J mice. LAT1 was stained with mLAT1 ( c ) antibody ( LAT1(Ms) ). Arrowheads indicate endothelial staining of LAT1. g Growth of B16-F10 tumors in Lat1 fl/fl and Lat1 fl/fl /Tek-Cre mice ( n = 4 each). h Gross appearance of tumors collected on Day 10. i Intratumoral blood vessels visualized by intravenously injected FITC-Dextran. j Quantification of the blood vessel area on tumor sections

Article Snippet: For western blotting using total membrane fractions of oocytes, following antibodies were used for the detection of LAT1 and 4F2hc: anti-mLAT1(R), anti-4F2hc (sc-7094, Santa Cruz Biotechnology), peroxidase-conjugated goat anti-rabbit IgG (111–035-003, Jackson ImmunoResearch), and peroxidase-conjugated mouse anti-goat IgG (205–035-108, Jackson ImmunoResearch).

Techniques: Inhibition, In Vivo, Control, Immunofluorescence, Immunohistochemistry, Staining, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Amino acid transporter LAT1 in tumor-associated vascular endothelium promotes angiogenesis by regulating cell proliferation and VEGF-A-dependent mTORC1 activation

doi: 10.1186/s13046-020-01762-0

Figure Lengend Snippet: Endothelial LAT1 expression induced by pro-angiogenic factors and its contribution to proliferation and translation regulation. a and b LAT1 mRNA expression in HUVECs starved for VEGF-A and FGF-2 followed by stimulation for 2 h with VEGF-A or FGF-2 alone (10 ng/mL), or with their combination ( a ), or for 24 h with VEGF-A and FGF-2 (10 ng/mL each) ( b ). c LAT1 protein expression in HUVECs starved as in ( a and b ), then stimulated with VEGF-A and FGF-2 (10 ng/mL each). Cell lysates were analyzed by western blotting. β-actin ; loading control. Bar graph ; densitometric quantification of the band intensity. d LAT1 knockdown (KD) in HUVECs. Crude membrane fractions prepared 48 h after transfection of control ( NC ) or LAT1-targeting siRNAs ( LAT1 siRNA #1–3 ) were examined by western blotting. Na + /K + -ATPase α 1 ; loading control. e and f Cell proliferation of LAT1 KD cells ( e ) and JPH203-treated cells ( f ). g and h Effects of LAT1 KD ( g ) and JPH203 ( h ) on mTORC1- and GAAC pathways. Cell lysates were prepared and analyzed 48 h after transfection of siRNAs ( g ), and after JPH203-treatment for 24 h ( h ). a , b , n = 4; c , n = 3; e , f , n = 8

Article Snippet: For western blotting using total membrane fractions of oocytes, following antibodies were used for the detection of LAT1 and 4F2hc: anti-mLAT1(R), anti-4F2hc (sc-7094, Santa Cruz Biotechnology), peroxidase-conjugated goat anti-rabbit IgG (111–035-003, Jackson ImmunoResearch), and peroxidase-conjugated mouse anti-goat IgG (205–035-108, Jackson ImmunoResearch).

Techniques: Expressing, Western Blot, Control, Knockdown, Membrane, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Amino acid transporter LAT1 in tumor-associated vascular endothelium promotes angiogenesis by regulating cell proliferation and VEGF-A-dependent mTORC1 activation

doi: 10.1186/s13046-020-01762-0

Figure Lengend Snippet: Suppression of migration, invasion and tube formation of endothelial cells by LAT1 knockdown and inhibition. a Wound healing assay using HUVECs transfected with control ( NC ) or LAT1-targeting siRNAs ( LAT1 siRNA #1–3 ). b Quantification of migrated cell numbers in assays using LAT1 KD cells ( left ) and cells treated with JPH203 (0, 1, 10, 50 μM; right ). c Invasion assay using LAT1 KD cells. d Quantification of covered surface areas in assays using LAT1 KD cells ( left ) and JPH203-treated cells ( right ). e Tube formation assay using LAT1 KD cells. f Quantification of total branching length of tubular network in assays using LAT1 KD cells ( left ), and JPH203-treated cells ( right ). b , d , n = 6; f , n = 7/8/8/4 for LAT1 KD, n = 8 for JPH203

Article Snippet: For western blotting using total membrane fractions of oocytes, following antibodies were used for the detection of LAT1 and 4F2hc: anti-mLAT1(R), anti-4F2hc (sc-7094, Santa Cruz Biotechnology), peroxidase-conjugated goat anti-rabbit IgG (111–035-003, Jackson ImmunoResearch), and peroxidase-conjugated mouse anti-goat IgG (205–035-108, Jackson ImmunoResearch).

Techniques: Migration, Knockdown, Inhibition, Wound Healing Assay, Transfection, Control, Invasion Assay, Tube Formation Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Amino acid transporter LAT1 in tumor-associated vascular endothelium promotes angiogenesis by regulating cell proliferation and VEGF-A-dependent mTORC1 activation

doi: 10.1186/s13046-020-01762-0

Figure Lengend Snippet: Expression of LAT1 in tumor-associated vascular endothelial cells. a Immunohistochemistry of LAT1 and CD31 in pancreatic ductal adenocarcinoma (PDA) and normal pancreas. Representative images from CU1372–35-35,006 (PDA) and CU2009/02 X-40 (normal pancreas) are shown. Arrows and arrowheads indicate tumor cells and endothelial cells, respectively. Black squares ; enlarged images. b Immunofluorescence of LAT1 ( green ) and CD31 ( purple ) in PDA and normal pancreas. Nuclei stained with DAPI (blue ) are shown in the merged images. c Endothelial LAT1 expression in tissue microarray containing PDA and normal pancreas. Tissues spots were classified depending on the LAT1 staining intensity in endothelial cells: High, Low/Medium, and Negative. Data shown are the percentage of each group. d and e Immunohistochemistry of mouse LAT1 and CD34 in paraffin sections of MIA PaCa-2 ( d ) and H520 ( e ) xenograft tumors. Arrowheads indicate endothelial cells. Black squares ; enlarged images. Mouse LAT1 was visualized with mouse-specific mLAT1( c ) antibody ( LAT1(Ms) )

Article Snippet: For western blotting using total membrane fractions of oocytes, following antibodies were used for the detection of LAT1 and 4F2hc: anti-mLAT1(R), anti-4F2hc (sc-7094, Santa Cruz Biotechnology), peroxidase-conjugated goat anti-rabbit IgG (111–035-003, Jackson ImmunoResearch), and peroxidase-conjugated mouse anti-goat IgG (205–035-108, Jackson ImmunoResearch).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Staining, Microarray

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Amino acid transporter LAT1 in tumor-associated vascular endothelium promotes angiogenesis by regulating cell proliferation and VEGF-A-dependent mTORC1 activation

doi: 10.1186/s13046-020-01762-0

Figure Lengend Snippet: Effects of LAT1 knockdown and inhibition on VEGF-A-dependent signaling pathways. a HUVECs starved for serum and growth factors were stimulated with VEGF-A (10 ng/mL) in the presence or absence of JPH203 (50 μM). b and c Effects of JPH203 and rapamycin, and LAT1 KD on mTORC1- and GAAC pathways under stimulation with VEGF-A. HUVECs starved for serum and growth factors were stimulated with VEGF-A (10 ng/mL) for 20 min in the presence or absence of JPH203 (50 μM) or rapamycin (10 nM) ( b ). HUVECs transfected with control siRNA ( NC ) or LAT1-targeting siRNA ( LAT1 siRNA #1 ) were starved and stimulated with VEGF-A (10 ng/mL) for 20 min ( c ). d Proposed molecular mechanism for the essential role of endothelial LAT1 in the VEGF-A-dependent activation of mTORC1. The LAT1-mediated amino acid signaling is independent of the PI3K-Akt axis in the downstream of VEGFR2, and is possibly mediated by Ragulator-Rag GTPase heterodimer complex that recruits inactive mTORC1 onto lysosomal surface for its interaction with kinase activator Rheb. The input of amino acid signaling is indispensable for the pro-angiogenic VEGF-A signaling to induce activation mTORC1 and subsequent angiogenesis

Article Snippet: For western blotting using total membrane fractions of oocytes, following antibodies were used for the detection of LAT1 and 4F2hc: anti-mLAT1(R), anti-4F2hc (sc-7094, Santa Cruz Biotechnology), peroxidase-conjugated goat anti-rabbit IgG (111–035-003, Jackson ImmunoResearch), and peroxidase-conjugated mouse anti-goat IgG (205–035-108, Jackson ImmunoResearch).

Techniques: Knockdown, Inhibition, Protein-Protein interactions, Transfection, Control, Activation Assay

Journal: Cell Death & Disease

Article Title: USP12 promotes breast cancer angiogenesis by maintaining midkine stability

doi: 10.1038/s41419-021-04102-y

Figure Lengend Snippet: A The quantitative proteomics of USP12-overexpressing and control HEK293T cells were analysed using the iTRAQ technique, and 36 DEPs (including 21 upregulated proteins and 15 downregulated proteins) were identified. B , C USP12, USP12 mutant (C48A) and control vectors were transfected into HEK293T cells, and the protein and mRNA levels of MDK and USP12 were detected by western blotting ( B ) and RT-qPCR ( C ). D USP12-Flag and the MDK-GST plasmid were co-transfected into HEK293T cells, and USP12-Flag was immunoprecipitated with anti-Flag antibody. E USP12-Flag and the MDK-GST plasmid were co-transfected into HEK293T cells, and MDK-GST was pulled down with glutathione-Sepharose 4B slurry beads. F Endogenous USP12 was captured by anti-MDK antibody from MDA-MB-231 cells, and the endogenous USP12 and MDK were examined by immunoblotting. G Endogenous USP12 and MDK expression in MDA-MB-231 (upper) and MCF7 (bottom panel) cells were detected by immunofluorescence staining. Scale bars, 30 μm.

Article Snippet: The antibodies used for Western blotting and IP in this study included anti-MDK antibody (sc-46701, Santa Cruz, USA), anti-USP12 antibody (LS-C370534, LSBio, USA), anti-CD31 antibody (77699, CST, USA), anti-Flag antibody (14793, CST, USA), anti-GST antibody (2624, CST, USA), anti-HA antibody (3724, CST, USA), mouse IgG (3420, CST), anti-GST agarose antibody (A8580, Sigma Aldrich) and anti-Flag ® M2 affinity gel (A2220, Sigma Aldrich).

Techniques: Mutagenesis, Transfection, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Immunoprecipitation, Expressing, Immunofluorescence, Staining

Journal: Cell Death & Disease

Article Title: USP12 promotes breast cancer angiogenesis by maintaining midkine stability

doi: 10.1038/s41419-021-04102-y

Figure Lengend Snippet: A MDK-Flag and HA-UB plasmids with USP12, USP12 mutant (C48A) and control vector were co-transfected into HEK293T cells for 36 h. After 6 h of incubation with 10 μM MG132, ubiquitination assay was performed to detect the poly-ubiquitination of MDK. B The endogenous poly-ubiquitination level of MDK in MDA-MB-231 cells after USP12 overexpression was detected by the deubiquitination assay. C Indicated plasmids were transfected into HEK293T cells; 36 h after transfection, the cells were treated with MG132 for 6 h and then subjected to ubiquitination assay. D USP12 was stably overexpressed in MDA-MB-231. Immunoblotting showed the protein levels of MDK and USP12. E USP12 was knocked down in MDA-MB-231 cells. Immunoblotting showed the protein levels of MDK and USP12. F USP12 was overexpressed in MDA-MB-231 cells, and the MDK level in the medium supernatant of MDA-MB-231 cells was detected by immunoblotting. G USP12 was knocked down in MDA-MB-231 cells, and the MDK level in the medium supernatant of MDA-MB-231 cells was detected by immunoblotting. H MDK was co-expressed with USP12, USP12 mutant (C48A) and control vector in HEK293T cells. After 36 h, cells were treated with CHX (50 µg/ml) for the indicated time intervals. The expression of MDK and USP12 was detected (left panel), and the intensity of MDK expression was quantified by ImageJ software (right panel). I USP12 was knocked down with shRNA in MDA-MB-23 cells, and the MDK half-life was analysed by CHX pulse-chase assay with immunoblotting (left panel) and quantified (right panel).

Article Snippet: The antibodies used for Western blotting and IP in this study included anti-MDK antibody (sc-46701, Santa Cruz, USA), anti-USP12 antibody (LS-C370534, LSBio, USA), anti-CD31 antibody (77699, CST, USA), anti-Flag antibody (14793, CST, USA), anti-GST antibody (2624, CST, USA), anti-HA antibody (3724, CST, USA), mouse IgG (3420, CST), anti-GST agarose antibody (A8580, Sigma Aldrich) and anti-Flag ® M2 affinity gel (A2220, Sigma Aldrich).

Techniques: Mutagenesis, Plasmid Preparation, Transfection, Incubation, Ubiquitin Assay, Over Expression, Stable Transfection, Western Blot, Expressing, Software, shRNA, Pulse Chase

Journal: Cell Death & Disease

Article Title: USP12 promotes breast cancer angiogenesis by maintaining midkine stability

doi: 10.1038/s41419-021-04102-y

Figure Lengend Snippet: A MDK and control vectors were transfected into MDA-MB-231 cells after USP12 knockdown, and immunoblotting showed the protein levels of MDK and USP12. B , C The angiogenesis of HUVECs was analysed by tube formation and migration assays ( B ). The tubes and migratory cells in panel B were quantified ( C ). D – I . The MDK and USP12 expression were analysed by immunoblotting MDK was knocked down in MDA-MB-231 USP12-overexpression cells ( D – F ) or overexpressed in MDA-MB-231-shUSP12 cells ( G – I ). The angiogenesis was analysed by the mouse aortic ring assay ( E , G ). Scale bars, 100 µm. J MDK and control vector were transfected into 4T1 cells after USP12 knockdown, and the protein levels of MDK and USP12 were detected by immunoblotting. K Lung metastasis nodules 5 weeks after the injection of 4T1 cells with USP12 knockdown and MDK overexpression into mouse mammary pads; the number of nodules in ( K ) was calculated ( L ). The data are presented as the mean ± SD, *** p < 0.001. M CD31 immunohistochemical staining of lung metastatic nodules in ( K ) is shown.

Article Snippet: The antibodies used for Western blotting and IP in this study included anti-MDK antibody (sc-46701, Santa Cruz, USA), anti-USP12 antibody (LS-C370534, LSBio, USA), anti-CD31 antibody (77699, CST, USA), anti-Flag antibody (14793, CST, USA), anti-GST antibody (2624, CST, USA), anti-HA antibody (3724, CST, USA), mouse IgG (3420, CST), anti-GST agarose antibody (A8580, Sigma Aldrich) and anti-Flag ® M2 affinity gel (A2220, Sigma Aldrich).

Techniques: Transfection, Western Blot, Migration, Expressing, Over Expression, Aortic Ring Assay, Plasmid Preparation, Injection, Immunohistochemical staining, Staining

Journal: Cell Death & Disease

Article Title: USP12 promotes breast cancer angiogenesis by maintaining midkine stability

doi: 10.1038/s41419-021-04102-y

Figure Lengend Snippet: A Representative staining of USP12 and MDK in breast cancer samples. Scale bars, 100 μm. B , C Comparison of the relative protein expression levels of USP12 and MDK in 32 paired breast cancer and adjacent normal tissues. * p < 0.05, *** p < 0.001; mean ± SD. D A positive correlation was observed between USP12 and MDK protein expression in breast cancer samples. E–H The relationship between USP12 or MDK expression and the OS or DMFS rate of breast cancer patients was analysed in the KM ( http://kmplot.com/analysis/ ) database.

Article Snippet: The antibodies used for Western blotting and IP in this study included anti-MDK antibody (sc-46701, Santa Cruz, USA), anti-USP12 antibody (LS-C370534, LSBio, USA), anti-CD31 antibody (77699, CST, USA), anti-Flag antibody (14793, CST, USA), anti-GST antibody (2624, CST, USA), anti-HA antibody (3724, CST, USA), mouse IgG (3420, CST), anti-GST agarose antibody (A8580, Sigma Aldrich) and anti-Flag ® M2 affinity gel (A2220, Sigma Aldrich).

Techniques: Staining, Expressing