Journal: Nature Communications

Article Title: Soluble E-cadherin promotes tumor angiogenesis and localizes to exosome surface

doi: 10.1038/s41467-018-04695-7

Figure Lengend Snippet: sE-cad-positive exosomes activate the NFκB pathway. a Top network identified by IPA. Gene signatures of HUVEC incubated with IgG pre-treated exosomes and HECD-1 pre-treated exosomes. b NFκB reporter assay of HUVEC treated with exosomes (25 μg mL −1 ) in the presence or absence of E-cadherin neutralizing antibodies, HECD-1 (100 μg mL −1 ) or VE-cadherin neutralizing antibodies, BV9 (10 μg mL −1 ). c Western blot analysis of the subcellular localization of NFκB subunits in HUVEC treated with exosomes (25 μg mL −1 ) in the presence or absence of E-cadherin neutralizing antibodies, HECD-1 (100 μg mL −1 ). d Tube formation assay of HUVEC treated with exosomes (25 μg mL −1 ) in the presence or absence of Bay11-7082 (Bay) (10 μM). Upper: Representative images of HUVEC tube formation assay. Lower: Quantification of the percentage change of the number of branching points. Bar, 100 μm. e Coimmunoprecipitation of NFκB subunits and β-catenin (β-cat) in HUVEC with or without exosomes treatment (25 μg mL −1 ). f NFκB reporter assay of HUVEC transiently transfected with non-specific (NS) siRNA or β-catenin (β-cat) siRNA (20 nM) for 24 h, treated with exosomes (25 μg mL −1 ). TCF reporter assay of HUVEC transiently transfected with NS siRNA or p105/p50 and p65 siRNA (20 nM) for 24 h, treated with exosomes (25 μg mL −1 ). g Western blot analysis of the β-catenin (β-cat) and NFκB subunits in HUVEC treated with exosomes (25 μg mL −1 ) for the indicated period of time. Upper: Representative images of nuclear fractions. Lower: Densitometry of β-catenin (β-cat) and p65. h Tube formation assay of HUVEC transiently transfected with non-specific (NS) siRNA, β-catenin siRNA, or p105/p50 siRNA and p65 siRNA (20 nM) for 24 h with or without exosomes treatment (25 μg mL −1 ). Upper: Representative images of HUVEC tube formation assay. Lower: Quantification of the percentage change of the number of branching points. Bar, 100 μm. n = 3 per group, all experiments were repeated three times. Error bar indicates SD of the mean. * P < 0.05 versus untreated control using one-way analysis of variance followed by Tukey’s least significant difference post hoc test

Article Snippet: Equal amounts of protein (1 mg) were precleared with protein A/G PLUS agarose beads (Santa Cruz Biotechnology) for 2 h. Supernatant was then incubated with anti-p65 antibodies or mouse IgG at 4 °C overnight.

Techniques: Incubation, Reporter Assay, Western Blot, Tube Formation Assay, HUVEC Tube Formation Assay, Transfection, Control