Journal: Scientific Reports

Article Title: CD146 is required for VEGF-C-induced lymphatic sprouting during lymphangiogenesis

doi: 10.1038/s41598-017-06637-7

Figure Lengend Snippet: CD146 is expressed in LEC and regulates LEC activation induced by VEGF-C. ( a ) WB analysis of CD146 and VEGFR-3 expression in four different endothelial cell lines. ( b ) Serum-starved HDLEC cells were treated with VEGF-C156S (100 ng/ml) for different time intervals. The expression of CD146 and VEGFR-3 was determined by WB. ( c – f ) HDLEC cells, transfected with siRNA of control, CD146, VEGFR-3 or combination of CD146 and VEGFR-3, were subjected to spheroid sprouting assay ( c ), proliferation assay (d), tube formation assay ( e ) and transwell migration assay ( f ). VEGF-C156S was applied at the indicated concentration. Data were expressed as means ± SEM from 3 independent experiments (n = 3 in each group of c – f ). Significant difference was determined by Student’s t- test (* P < 0.05; ** P < 0.01; *** P < 0.001; ns, no significant difference). Full-length blots are presented in Supplementary Fig. .

Article Snippet: The following reagents were used: recombinant human VEGFR-3-Fc, human VEGF-C, Fc-CD146 and sCD146 were from Sino Biological.

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

Journal: Scientific Reports

Article Title: CD146 is required for VEGF-C-induced lymphatic sprouting during lymphangiogenesis

doi: 10.1038/s41598-017-06637-7

Figure Lengend Snippet: VEGF-C elicitation of different signaling cascades downstream of CD146 or VEGFR-3. ( a , b ) Dose and time effect of VEGF-C156S on HDLEC cells. Serum-starved cells were treated with VEGF-C156S for 20 min at different concentrations in ( a ) or with 100 ng/ml of VEGF-C156S at different time intervals in ( b ). Dimerization of CD146 and phosphorylation and expression of AKT, ERK and p38, elicited from VEGF-C156S, were analyzed by WB. ( c ) HDLEC cells, transfected with siRNA of control, CD146, VEGFR-3 or combination of CD146 and VEGFR-3, were treated with 100 ng/ml of VEGF-C156S for 20 min. ( d – g ) HDLEC cells, pretreated with SCH772984 or FHPI for 72 hr, were subjected to spheroid sprouting assay ( d ), proliferation assay ( e ), tube formation assay ( f ) and transwell migration assay ( g ). VEGF-C156S was applied at the indicated concentration. Data were expressed as means ± SEM from 3 independent experiments (n = 12 in each group of d – g ). Significant difference was determined by Student ’ s t- test (* P < 0.05; ** P < 0.01; *** P < 0.001; ns, no significant difference). Full-length blots are presented in Supplementary Fig. .

Article Snippet: The following reagents were used: recombinant human VEGFR-3-Fc, human VEGF-C, Fc-CD146 and sCD146 were from Sino Biological.

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

Journal: Scientific Reports

Article Title: CD146 is required for VEGF-C-induced lymphatic sprouting during lymphangiogenesis

doi: 10.1038/s41598-017-06637-7

Figure Lengend Snippet: CD146 directly binds to VEGF-C. ( a ) Co-immunoprecipitation assays in HDLECs. Cells were incubated with normal culture medium or VEGF-C156S (100 ng/ml) conditional medium. The cell lysates were prepared for immunoprecipitation with control mIgG or anti-CD146 mAb AA1. ( b , d ) HEK293 cells transfected with control empty or CD146 expression vectors were incubated with VEGF-C conditional medium or transfected with CD146 encoding vector without VEGF-C incubation. Binding of VEGF-C-His to cells was detected by co-immunoprecipitation ( b ) and immunofluorescence ( d ). Scale bars: 100 μm. ( c ) Direct in vitro interaction between VEGF-C and CD146 proteins. Purified Fc, Fc-CD146 or Fc-VEGFR-3 (200 ng/ml) was incubated with VEGF-C protein (200 ng/ml). The results were analyzed by WB. ( e , f ) defining the domain of CD146 required for interaction with VEGF-C. CD146 and its truncation mutants were expressed as Flag-tagged proteins. Immunoprecipitation was performed using anti-VEGF-C mAb. Full-length blots are presented in Supplementary Fig. .

Article Snippet: The following reagents were used: recombinant human VEGFR-3-Fc, human VEGF-C, Fc-CD146 and sCD146 were from Sino Biological.

Techniques: Immunoprecipitation, Incubation, Transfection, Expressing, Plasmid Preparation, Binding Assay, Immunofluorescence, In Vitro, Purification

Journal: Scientific Reports

Article Title: CD146 is required for VEGF-C-induced lymphatic sprouting during lymphangiogenesis

doi: 10.1038/s41598-017-06637-7

Figure Lengend Snippet: Functional cytoplasmic domain of CD146 in VEGF-C induced cell activation. ( a ) Diagrammatic representation of C-terminal truncations of CD146 at the intracellular domain. ( b ) Phosphorylation and expression of ERK1/2 and p38, elicited from VEGF-C156S, were analyzed by WB. HEK293 cells were transfected with plasmids encoding truncated versions of CD146 as shown in ( a ). ( c – g ) HDLECs transfected with plasmids encoding CD146-WT and CD146-∆599-646 were subjected to signaling activation assay ( c ), spheroid sprouting assay ( d ), proliferation assay ( e ), tube formation assay ( f ) and transwell migration assay ( g ). VEGF-C was applied at the indicated concentration. Data were expressed as means ± SEM from 3 independent experiments (n = 12 in each group of c – g ,). Significant difference was determined by Student’s t- test (* P < 0.05; ** P < 0.01; *** P < 0.001; ns, no significant difference). Full-length blots are presented in Supplementary Fig. .

Article Snippet: The following reagents were used: recombinant human VEGFR-3-Fc, human VEGF-C, Fc-CD146 and sCD146 were from Sino Biological.

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

Journal: Scientific Reports

Article Title: CD146 is required for VEGF-C-induced lymphatic sprouting during lymphangiogenesis

doi: 10.1038/s41598-017-06637-7

Figure Lengend Snippet: CD146 modulates VEGF-C induced lymphatic development in zebrafish. ( a ) CD146 expression in the zebrafish embryos at 5 dpf was detected by whole-mount in situ hybridization (WISH). lyve1b was used as positive control. Black arrows indicate the position of TD. DA, dorsal aorta; PCV, posterior cardinal vein; and TD, thoracic duct. Scale bars: 30 μm. ( b ) The effect of knockdown cd146 or vegfc on blood vessels. Bright-field lateral views of the trunk of embryos injected with control, cd146 or vegfc specific MOs. Embryos were stained by WISH for a panel of arterial markers of dll4 , tbx20 and ephrinB2 and venous markers of msr and flt4 . Black arrows, blood vessels; DA, dorsal aorta; and PCV, posterior cardinal vein. Scale bars: 125 μm. ( c ) The effect of knockdown cd146 or vegfc on vasculature of blood and lymph. The control, cd146 or vegfc specific MO were injected into the transgenic zebrafish Tg ( fli1a : EGFP) embryos, respectively. The embryos harvested at 36 hpf, 48 hpf and 72 hpf were analyzed. The confocal images were flanked by redrawing of the vessel contours. Transient lymphangiogenic structures (lymphangiogenic sprouts; PL cells) were labeled with light green. The other vessels (PCV, DA and ISV) were labeled with dark grey. Scale bars: 20 μm. ( d ) The effects of cd146 knockdown on development of lymphatic capillary. The control or cd146 specific MO was co-injected with control or human vegfc mRNA into the transgenic zebrafish Tg ( fli1a : EGFP) embryos. Late-phase microangiography was used to visualize lymphatic capillaries in embryos at 3 dpf. Images showed the representative phenotypes of each group and the ratio indicated the embryos with such representative phenotype over the tested embryos. Scale bars: 50 μm.

Article Snippet: The following reagents were used: recombinant human VEGFR-3-Fc, human VEGF-C, Fc-CD146 and sCD146 were from Sino Biological.

Techniques: Expressing, In Situ Hybridization, Positive Control, Injection, Staining, Transgenic Assay, Labeling

Journal: World Journal of Gastroenterology

Article Title: Role of VEGF and CD44v6 in differentiating benign from malignant ascites

doi: 10.3748/wjg.v9.i11.2596

Figure Lengend Snippet: Comparison of VEGF concentrations in different kinds of ascites. Group 1: cirrhotic ascites, Group 2: tuberculous ascites, Group 3: malignant ascites.

Article Snippet: Immunoassay for human VEGF Concentrations of VEGF in ascites were determined with an ELISA kit (R&D Systems) following the manufacturer’s guidelines.

Techniques: Comparison