Schematic representation of signaling cascade involved in hypoxia-inducible ephrin-A1 modulation of angiogenesis in tumor hypoxic microenvironment.

<p>Schematic representation of signaling cascade involved in hypoxia-inducible ephrin-A1 modulation of angiogenesis in tumor hypoxic microenvironment.</p

Effect of ephrin-A1-Fc on expression and phosphorylation of eNOS and Akt in HUVECs. HUVECs were exposed to ephrin-A1-Fc (1 µg/ml) for 0 h, 8 h, 24 h.

<p>Immunofluorescence (A), Real-time PCR (B) and Western blot analysis (C) demonstrated that ephrin-A1-Fc had no effect on eNOS expression of HUVECs (#, <i>P</i>>0.05, n = 3). D: Western blots demonstrating that P-eNOS_Ser1177 and P-Akt_Ser473 were up-regulated under ephrin-A1-Fc stimulation in a time-dependent manner. E: Quantity analysis of P-eNOS_Ser1177. F: Quantity analysis of P-Akt_Ser473. (*, <i>P</i><0.05, n = 4).</p

NO production in the supernatants of HUVECs after incubation with ephrin-A1-Fc (1 µg/ml) for 0 h, 24 h.

<p>Data showed a significantly increased NO production in the EA1-Fc group. (*, <i>P</i><0.05, n = 3).</p

Hypoxia up-regulated ephrin-A1 expression and soluble ephrin-A1 secretion in cancer cells.

<p>A: Western blots demonstrating that hypoxia elevated membrane bound ephrin-A1 expression in SCC-9 cells. B: Western blots demonstrating that hypoxia up-regulated soluble ephrin-A1 in supernatants of SCC-9 cells. SCC-9 cell density at 70–80% confluence was taken as 0 h when fresh culture medium was added. C: Cell rounding assay demonstrating that soluble ephrin-A1 in CM could activate EphA2 in U-251 GBM cells. Ephrin-A1(S), soluble ephrin-A1; N, normoxia conditioned medium group; H, hypoxia conditioned medium group.</p

PI3K/Akt mediated ephrin-A1-induced P-eNOS_Ser1177 in HUVECs.

<p>A: Representative Western blots for P-eNOS_Ser1177 and P-Akt_Ser473 from HUVECs that were starved in 0.1%BSA EBM-2 overnight and stimulated with ephrin-A1-Fc (1 µg/ml) for 30 min alone or together pre-treated with LY294002. B: Quantity analysis of P-Akt_Ser473. C: Quantity analysis of P-eNOS_Ser1177. (*, <i>P</i><0.05, n = 4) (#, <i>P</i>>0.05, n = 4).</p