Fig. 11

NP-G2-044 inhibited FSCN1 binding to F-actin, resulting in reduced nuclear entry of YAP and inhibited CDC42 GTP activity. A Western blotting assays test the protein expression of p-VEGFR2(Tyr1175), VEGFR2, CDC42-GTP, CDC42-Total, p − YAP(Ser127), YAP and GAPDH in HRMECs after treatment with VEGF-A (20 ng/ml) for 5 min and different concentrations of NP − G2 − 044. (n = 4 independent experiments). B Densitometric quantitation of Western blot band intensity shown in A. Results are presented as mean ± SEM, statistical analyses were performed using One-way ANOVA. (n = 4 independent experiments). C HRMECs were transfected with OE-NC, OE-FSCN1, OE-FSCN1 added with 1 μM NP − G2 − 044 or left untreated for 24 h. The group without any treatment is taken as the control group (D, F). Total, cytoplasmic, nuclear extracts from the resulting cells are analyzed by WB for YAP expression. (n = 4 independent experiments). D Densitometric quantitation of Western blot band intensity shown in C. Results are presented as mean ± SEM, statistical analyses were performed using One-way ANOVA with Bonferroni's post hoc test. (n = 4 independent experiments). E Localization of YAP is demonstrated by immunofluorescence. Scale bar, 50 μm. (n = 5 per group, data pooled from 5 independent experiments). F HRMECs were transfected with OE-NC, OE-FSCN1, OE-FSCN1 added with 1 μM NP − G2 − 044 or left untreated in the presence or absence of 5 μM verteporfin for 24 h, DMSO as a vehicle control. Three-dimensional (3D) Bead Sprouting Assay reveals the in vitro sprouting capabilities of HRMECs under different treatments. Scale bar: 100 µm. (n = 4 per group, data pooled from 4 independent experiments). G Quantification of the rate of Nuclear/Total YAP fluorescence. Results are presented as mean ± SEM, statistical analyses were performed using Kruskal–Wallis with Bonferroni's post hoc test. (n = 5 per group, data pooled from 5 independent experiments). H, I Quantification of length and number of sprouts per bead. Results are presented as mean ± SEM, statistical analyses were performed using One-way ANOVA with Bonferroni's post hoc test. (n = 4 per group, data pooled from 4 independent experiments)