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. 2014 Mar 22;8(5):894–911. doi: 10.1016/j.molonc.2014.03.011

Figure 9.

Figure 9

β‐catenin‐14‐3‐3ζ extracellular vesicles activate the Wnt pathway. (A) HEK293T transfected with the pTOPFLASH reporter vectors and β‐gal were incubated with increasing amounts of purified extracellular vesicles (derived from double transfected cells as described above). Relative luciferase values are shown. (B) Luciferase assay was performed in HeLa, Huh7 and COS‐7 acceptor cells (incubated with purified extracellular vesicles – as described above) transfected with the pTOPFLASH reporter system and renilla CMV. Relative luciferase values are shown. (C) Purified extracellular vesicles were resuspended in PBS and then added to 2 × 106/per 60 mm plate HEK293‐EBNA‐PurR acceptor cells (Skalka et al., 2013). Twenty four hours later, the growth media was replaced with puromycin CM (1 μg/ml) or with medium collected from L‐wnt3a cells. Thirty h later the cells were fixed and stained using methylene blue. (D) Extracellular vesicles from HEK293T cells (left panel) or SW480 (right panel) do not contain the Wnt3a ligand. Growing medium from L‐Wnt3a cells served as a positive control. Cell lysates or extracellular vesicles were incubated with the indicated antibodies.