Journal: Nature Communications
Article Title: A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis
Figure Lengend Snippet: VEGF released from MVs regains its sensitivity to Bevacizumab. ( a ) Non-permeabilized MDAMB231 cells were analysed by immunofluorescent confocal microscopy using anti-VEGF and anti-Hsp90 antibodies. Top images: VEGF 90K and Hsp90 are detected on MVs (arrows). Bottom images: MDAMB231 cells treated with 10 μM 17AAG overnight were fixed and stained. Scale bar, 10 μm. Far-right: Blow-ups of the MVs. ( b ) MDAMB231 cell MVs treated without (lane 2) or with (lane 3) 17AAG at 37 °C for 2 h were lysed and immunoprecipitations were performed using a Hsp90 antibody (25 μg MV protein, each). ( c ) VEGF 90K was generated by tTG-catalysed crosslinking of rVEGF 165 , incubated with Hsp90 (30 ng), either without (lane 1) or with 17AAG (10 μM) (lane 2), at 37 °C for 1 h and immunoprecipitated using an anti-Hsp90 antibody. ( d ) MDAMB231 cell MVs treated without (lane 1) or with 17AAG (lane 2) at 37 °C for 2 h were collected on a 0.22 μm filter. The filtered MVs and the flow-through were immunoblotted. ( e ) Serum-deprived HUVECs were untreated (lane 1), or exposed to VEGF 90K (∼10 ng ml −1 ) released from 17AAG-treated MVs and present in the flow-through from the experiment shown in Fig. 5d , in the presence (lane 2) or absence (lane 3) of Bevacizumab, for 15 min, lysed and immunoblotted. ( f ) Serum-starved HUVECs, untreated (lanes 1, 2, 5 and 6) or pre-treated with 10 μM GA for 1 hour (lanes 3 and 4), were incubated without (lane 1) or with MVs (5 μg ml −1 protein) from MDAMB231 cells (lane 2), together with 200 ng ml −1 pan inactivating VEGF antibody (lane 4), 10 μM 17AAG (lane 5) or both the VEGF antibody and 17AAG (lane 6), for 15 min. Cell extracts were lysed and immunoblotted. ( g ) Serum-starved HUVECs were incubated with the VEGF 90K –Hsp90 complex (∼10 ng ml −1 ) without (lane 1) or with Bevacizumab (0.5 μg ml −1 ) (lane 2) for 15 min, lysed and immunoblotted. ( h ) Left: Relative amounts of tubulogenesis for HUVECs untreated (control; histogram 1) or treated with the VEGF 90K –Hsp90 complex (∼20 ng ml −1 total protein) without (histogram 3) or with 0.5 μg ml −1 Bevacizumab (histogram 4). Right: Images of the tubulogenesis assays.
Article Snippet: Reagents The pan VEGF antibody that recognizes all forms of VEGF-A was obtained from Santa Cruz (Dallas, TX, USA) (SC-507; used at 1:2,000).
Techniques: Confocal Microscopy, Staining, Generated, Incubation, Immunoprecipitation, Flow Cytometry