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. 2017 Jun 6;8:15729. doi: 10.1038/ncomms15728

Figure 6. sEV-associated EphA2 promotes cancer cell proliferation through EphA2/ephrin-A1 reverse signalling.

Figure 6

(a) Immunoblotting of EphA2 and β-actin in the WCL of MCF-7 cells expressing empty vector or ectopic EphA2. Cells were grown for 3 days with or without CM prepared from DXR-induced senescent RPE-1 cells. (b) EphA2 immunoprecipitates prepared from MCF-7 cells expressing ectopic EphA2 were immunoblotted with anti-phosphotyrosine and anti-EphA2 antibody. The indicated samples were treated with recombinant human ephrin-A1 (500 ng ml−1) for 20 min. Eight-hour pre-treatment with the EphA2 inhibitor ALW-II-41-27 (100 nM) suppressed ephrin-A1-induced EphA2 phosphorylation. (c) Relative numbers of MCF-7 cells grown for 3 days in the presence of CM compared with the number of cells grown for 3 days in normal medium. DMSO or ALW-II-41-27 (100 nM) was added to the medium. (d) Immunoblotting of phospho-Erk, total-Erk and β-actin in the WCL of MCF-7 cells grown for 3 days with or without CM prepared from control or DXR-induced senescent RPE-1 cells expressing non-targeting shRNA (shNT), shEphA2 or shRab35. (e) The heatmap shows the relative gene expression levels in MCF-7 cells grown for 3 days with or without CM prepared from DXR-induced senescent RPE-1 cells expressing shNT or shEphA2. Genes whose expression levels were changed more than 1.2-fold by the CM of shNT-expressing cells are shown. (f) EphA2-dependent upregulated genes were analysed with oPOSSUM-3. Each circle in the plot shows an enrichment of the targets of a different transcription factor. (g) Immunoblotting of phospho-Erk, total-Erk and β-actin in the WCL of MCF-7 cells grown for 3 days with or without CM. Dot plot represents the number of MCF-7 cells grown for 3 days in the presence of CM compared with the number of cells grown for 3 days in normal medium. DMSO or U0126 (500 nM) was added to the medium. CM was prepared from DXR-induced senescent RPE-1 cells. Replicates are biological replicates (n=3). Error bars indicate s.d. *P<0.05 (two-tailed t-test).