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. 2020 Dec 8;6:140. doi: 10.1038/s41420-020-00378-9

Fig. 3. FN-EDA promoted phosphorylation of VEGFR2 in vivo and vitro.

Fig. 3

A Immunoblot result of pVEGFR2 and total VEGFR2 in AAV9-FN-EDA-shRNA or AAV9-Ctrl infected mice with experimental hepatic fibrosis (n = 3). (Data are shown as mean ± SEM; *P < 0.01.) B, C Immunoblot result of pVEGFR2 in HUVECs and HSECs co-cultured with FN-EDA-siRNAs or Ctrl-siRNA transfected LX-2 cells for 8 h (n = 3). (Data are shown as mean ± SEM; HUVEC: EDA-siRNA1 versus Ctrl, ***P < 0.0001; EDA-siRNA2 versus Ctrl, **P < 0.0001; HSEC: EDA-siRNA1 versus Ctrl, *P < 0.01; EDA-siRNA2 versus Ctrl, *P < 0.01.) D, E Immunoblot result of pVEGFR2 in HUVECs and HSECs co-cultured with LX-2 for 8 h. LX-2 were pretreated with neutralizing antibodies IST-9 or 3E2 or mouse IgG (40 μg/ml) for 0.5 h (n = 3). (Data are shown as mean ± SEM; HUVEC: IST-9 versus Ctrl, *P < 0.01; 3E2 versus Ctrl, *P < 0.01; HSEC: IST-9 versus Ctrl, **P < 0.001; 3E2 versus Ctrl, **P < 0.001.) F Tube formation assay of HUVECs treated with pFN, FN-EDA or rEDA (40 μg/ml) for 6 h (n = 3). (Data are shown as mean ± SEM; rEDA versus Ctrl: *P < 0.01; FN-EDA versus Ctrl: **P < 0.001; pFN versus Ctrl: ns. Scale bar = 100 μm.) G Migration assay of HUVECs treated with pFN, FN-EDA or rEDA (40 μg/ml) for 24 h. (Data are shown as mean ± SEM, n = 3. rEDA versus Ctrl: ****P < 0.00001; FN-EDA versus Ctrl: ****P < 0.00001; pFN versus Ctrl: **P < 0.001. Scale bar = 100 μm.) H Tube formation assay of HSECs treated with pFN, FN-EDA or rEDA (40 μg/ml) for 6 h (n = 3). (Data are shown as mean ± SEM; rEDA versus Ctrl: **P < 0.001; FN-EDA versus Ctrl: **P < 0.001; pFN versus Ctrl: ns. Scale bar = 100 μm.) I Migration assay of HSECs treated with pFN, FN-EDA or rEDA (40 μg/ml) for 24 h. FN-EDA and rEDA significantly promote HSECs tube formation (n = 3). (Data are shown as mean ± SEM; rEDA versus Ctrl: *P < 0.01; FN-EDA versus Ctrl: **P < 0.001; pFN versus Ctrl: ns. Scale bar =100 μm.) J Immunoblot result of VEGFR2 related pathway in HUVECs treated with pFN, FN-EDA or rEDA (40 μg/ml) for 6 h. (Data are shown as mean ± SEM; pVEGFR2: rEDA versus Ctrl: *P < 0.01; FN-EDA versus Ctrl: ***P < 0.0001; pFN versus Ctrl: ns; pPLCγ rEDA versus Ctrl: **P < 0.001; FN-EDA versus Ctrl: **P < 0.001; pFN versus Ctrl: ns; pPI3K rEDA versus Ctrl: ***P < 0.0001; FN-EDA versus Ctrl: ***P < 0.0001; pFN versus Ctrl: ns; pAKT rEDA versus Ctrl: *P < 0.01; FN-EDA versus Ctrl: ***P < 0.0001; pFN versus Ctrl: *P < 0.01; pERK rEDA versus Ctrl: *P < 0.01; FN-EDA versus Ctrl: **P < 0.001; pFN versus Ctrl: ns.) K Immunoblot result of pVEGFR2 in HUVECs. HUVECs were treated with FN-EDA (40 μg/ml) and/or ZM323881 (5 μM) for 6 h (n = 3). (Data are shown as mean ± SEM; FN-EDA versus FN-EDA + ZM323881, **P < 0.001; FN-EDA + ZM versus ZM323881, ****P < 0.00001).