Activating CD137 Signaling Promotes Sprouting Angiogenesis via Increased VEGFA Secretion and the VEGFR2/Akt/eNOS Pathway

Figure 4

Inhibition of the VEGFR2/Akt/eNOS pathway attenuated CD137 signaling activation-mediated endothelial function. (a, b) The relative protein expression of p-Akt(Ser473) after treatment with different concentrations of LY294002 (0, 2.5, 5, 10, 25, 50, and 75 μM) for 30 minutes and analyzed to β-actin. c Detection of p-VEGFR2 (Tyr1173), p-Akt (Ser473), and p-eNOS (Ser1177) proteins by western blot after treatment with VEGFR2 siRNA or/and LY294002 then activation of CD137 signaling for 10 minutes. The protein relative expression was quantified to total proteins (d). (i) The tube formation of HUVECs after treatment with VEGFR2 siRNA or/and LY294002 with the CD137 signaling activation. (e, f). (j, k) the EdU-555 proliferation assay and transwell assay were applied to detect endothelial proliferative and migratory ability. (j, k) Red and blue fluorescence represent the proliferating cells and nuclei, respectively, and the violet color represents migrating cells stained with 0.1% crystal violet. (g, h) The numbers of proliferative and migratory ECs were analyzed with Image-Pro Plus 8.0. . All treatments were under the circumstance of the CD137 signaling activation. , .

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