Fig. 4

NO-induced activation of FAK/Erk1/2-MAPK signaling pathway contributed to enhanced pro-angiogenic properties of MG-CM. a Western blot was employed to detect the phosphorylation levels of FAK and Erk1/2. b The phosphorylation level of Erk1/2 was examined after Erk1/2-MAPK-selective inhibitor PD98059 application for 4 h. c, d Tube formation assay was performed to detect the angiogenic abilities of HUVECs when PD98059 was added or not, followed by quantitative analysis. e Immunofluorescence staining results showed the expression of Ki67 and CD31 in different groups. DAPI was for nuclear counterstain. f The mRNA expression level of VEGF was detected by qRT-PCR, and GAPDH was used as internal reference. g The schematic illustration of MG-induced HIF-1α/eNOS/NO activation of EPCs promoting HUVECs’ proliferation, migration, and angiogenesis. This experiment was repeated independently three times. Abbreviations: EPCs, endothelial progenitor cells; HUVECs, human umbilical vein endothelial cells; FAK, focal adhesion kinase; NG-CM, conditioned media from endothelial progenitor cells under normal gravity; MG-CM, conditioned media from endothelial progenitor cells under microgravity; NO, nitric oxide; DAPI, 4, 6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; VEGF, vascular endothelial growth factor; HIF-1α, hypoxia-induced factor-1α; eNOS, endothelial nitric oxide synthase. Data were presented as the mean ± standard deviation. *P < 0.05, **P < 0.01, and *** P < 0.001