Figure 3. MVs shed from breast cancer cells contain an oligomeric VEGF species.

(a) Whole cell lysates (WCL) from MDAMB231 cells (lane 1) and human recombinant VEGF₁₆₅ (rVEGF₁₆₅; lane 2) were immunoblotted with antibodies against VEGF₁₆₅. (b) Concentrated conditioned medium (20 μg total protein) from serum-starved MDAMB231 (lane 1), HeLa (lane 2) or SKBR3 cells (lane 3) were immunoblotted. (c) MDAMB231 cells were analysed by immunofluorescent (IF) microscopy using Rhodamine-conjugated phalloidin (top panels) and either a pan VEGF or anti-VEGF₁₆₅ antibody (middle panels). Arrows indicate VEGF localized on MVs. Scale bar, 2 μm. IF images of MDAMB231 cells stained by secondary antibody (control; bottom panels). Scale bar, 10 μm (d) Exosomes (lane 1) or MVs (lane 2) from MDAMB231 cells were isolated, lysed and immunoblotted (5 μg per samples) with antibodies against VEGF, CD-63, actin and flotillin-2. (e) MVs from SKBR3 (lane 2), HeLa (lane 3) or MDAMB231 (lane 4) cells were isolated and lysed. WCL from MDAMB231 cells (lane 1), as well as MV lysates (10 μg per sample), were immunoblotted with antibodies against VEGF₁₆₅, flotillin-2 and the cytosolic-specific marker IκBα. (f) MVs from MDAMB231 cells transfected with control siRNA (lane 1) or siRNAs targeting VEGF (lanes 2–4) were immunoblotted with a pan VEGF antibody or anti-flotillin-2. WCL were immunoblotted with a pan VEGF antibody and an anti-actin antibody. (g) Tubulogenesis assays on HUVECs that were untreated (control) or treated with MVs from MDAMB231 cells (10 μg ml⁻¹ MV protein) transfected with control siRNA or siRNAs targeting VEGF. (h) MVs from MDAMB231 cells transfected with control siRNA (lane 1) or siRNAs targeting tTG (lanes 2 and 3) or treated with MDC (50 μM) (lane 4) were immunoblotted with antibodies against VEGF or flotillin-2, while WCLs were immunoblotted with antibodies against VEGF, tTG or actin. (i) Tubulogenesis assays of HUVECs treated with MVs (10 μg ml⁻¹ MV protein) from MDAMB231 cells expressing control siRNA, siRNAs targeting tTG or with MVs from MDAMB231 cells treated with 50 μM MDC.