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Fig. 3 | Molecular Cancer

Fig. 3

From: Exosome-mediated microRNA signaling from breast cancer cells is altered by the anti-angiogenesis agent docosahexaenoic acid (DHA)

Fig. 3

DHA increases exosome secretion from breast cancer cells and reduces tube formation by endothelial cells without affecting VEGF secretion. a Western blot analysis of CD63 expression in exosomes isolated from MCF7 and MDA-MB-231 cells after treatment with DHA. Equal volumes of exosome protein lysate were loaded and SDS-PAGE was run under non-reducing conditions. b CD63-GFP tagged exosomes secreted into cell culture medium from DHA-treated or untreated MCF7 and MDA-MB-231 breast cancer cells were isolated by ultracentrifugation, re-suspended in PBS and the GFP levels were measured by fluorescent spectrometry, * p < 0.0001 using one-way ANOVA (n = 3). c MCF7 and MDA-MB-231 breast cancer cells were treated with 100 μM DHA for 24 h. The media was removed for exosome isolation and the breast cancer cells that remained after DHA treatment were counted and are expressed as the average cell number per 106 cells (n = 3, error bars = SE). d MCF7 cells were treated with 100 μM DHA for 24 h, the exosomes from the treated and untreated cultures were isolated from the culture media by ultracentrifugation and stained with acridine orange (AO). The AO-labeled exosomes (100 μg) were incubated with EA.hy926 cells (1×104) seeded on ECMatrix in a 12-well plate for 24 h and imaged with a Perkin Elmer Operetta at 40× magnification, bright field (left) and fluorescence (right), 460 nm excitation and 650 nm emission. e Approximately 1/10 of the exosomes isolated from a confluent 100 mm plate of DHA-treated or untreated MCF7 cells were mixed with 1×104 EA.hy926 cells and seeded on ECMatrix in a 96-well plate and incubated for 18 h. Tube formation was visualized with a light microscope (lower) and counted (upper) *, p < 0.001 using one-way ANOVA (n = 4). f MCF7 cells were treated with 100 μM DHA, 1 mM clofibrate (CF) or 20 μM troglitazine (Trog) for 24 h and VEGF in the medium was analyzed by ELISA and expressed as pg/ml. *, p < 0.001 using one-way ANOVA (n = 4-6)

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