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. 2018 Jan 24;8:1539. doi: 10.1038/s41598-018-19788-y

Figure 1.

Figure 1

Increased expression of ephrin-A1 in BCa cells and HUVECs co-cultures compared to SV-HUC-1 cells and HUVECs co-culture. BCa cells (RT4, T24, TCCSUP cells) were co-cultured with HUVECs for 48 h, respectively. (A,B) Real-time PCR (A; n = 3) and western blot analysis (B; n = 3) respectively showed significant up-regulation of ephrin-A1 mRNA and protein expression in BCa cells (n = 3, respectively) co-cultured compared to that in SV-HUC-1 cells (n = 3; *p < 0.05, **p < 0.01). (C,D) ELISA (C; n = 3) showed significantly enhanced expression of sEphrin-A1 in the supernatants of BCa cells and HUVECs co-cultures (n = 3, respectively) compared to those from SV-HUC-1 and HUVECs co-culture (n = 3; *p < 0.05, **p < 0.01), which was further verified by western blot assay (D; n = 3, respectively). The increase in ephrin-A1 expression was highest in TCCSUP cells, followed by T24 cells and RT4 cells. (E) Western blot analysis demonstrated significant down-regulation of EphA2 protein expression in HUVECs from the lower chambers of BCa cell and HUVEC co-cultures (n = 3) compared to that in HUVECs from SV-HUC-1 and HUVEC co-cultures (n = 3; *p < 0.05, **p < 0.01). The changing trends of EphA2 protein expression in HUVECs were contrary to ephrin-A1 expression in BCa cells; down-regulation was lowest in HUVECs co-cultured with TCCSUP cells, slightly higher in those co-cultured with T24 cells and highest in those co-cultured with RT4 cells. (F,G) Transwell assay (F; n = 3) and tube formation test (G; n = 3) demonstrated significant up-regulation in migration and capillary-like structure formation of HUVECs respectively under treatment of supernatants from BCa cells and HUVECs co-cultures (n = 3, respectively) compared to that from SV-HUC-1 cells and HUVECs co-culture (n = 3; *p < 0.05, **p < 0.01). (H) Ex vo aortic ring angiogenesis assay showed similar changes in transwell assays and tube formation tests (n = 3, respectively; *p < 0.05, **p < 0.01). The results of vascular functional experiments demonstrated that vascularity was highest under treatment of supernatants from TCCSUP cell and HUVEC co-cultures, moderate in those from T24 cell and HUVEC co-cultures, and lowest in those from RT4 cell and HUVEC co-cultures, consistent with the up-regulation of sEphrin-A1 expression in supernatants. Original magnification = ×5. Results are expressed as the mean ± S.E.M.