Fig. 9

AFAP1L1 regulates the angiogenic activity of ECs via the YAP-DLL4-NOTCH axis. A HUVECs were transfected with shNC, shAFAP1L1 #1, shYAP or simultaneously added with DAPT for 24 h. Three-dimensional (3D) Bead Sprouting Assay reveals the in vitro sprouting capabilities of HUVECs under different treatments. Scale bar: 100 µm. 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 independent experiments). B Western blot analysis of DLL4, NICD, HES1, HES1 and β-actin protein levels in HUVEC cells transfected with shNC, shAFAP1L1 #1, shYAP or simultaneously added with DAPT for 24 h. Densitometric quantitation of western blot band intensity shown in B. Results are presented as mean ± SEM, statistical analyses were performed using one-way ANOVA with Bonferroni's post hoc test. (n = 4 independent experiments). C HUVECs were transfected with Vector, OE AFAP1L1 #1, shYAP or added with VP for 24 h. Three-dimensional (3D) Bead Sprouting Assay reveals the in vitro sprouting capabilities of HUVECs under different treatments. Scale bar: 100 µm. 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 independent experiments). D Western blot analysis of p-YAP(Ser127), YAP, DLL4, NICD and β-actin protein levels in HUVEC cells transfected with Vector, OE AFAP1L1, shYAP or simultaneously added with VP for 24 h. Densitometric quantitation of western blot band intensity shown in D. 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 HUVECs were transfected with Vector, OE AFAP1L1 or simultaneously transfected with shAFAP1L1 #1 or shAFAP1L1 #2. Total, cytoplasmic, nuclear extracts from the resulting cells are analyzed by western blot for YAP expression. Densitometric quantitation of western blot band intensity shown in E Results are presented as mean ± SEM, statistical analyses were performed using one-way ANOVA with Bonferroni's post hoc test. (n = 4 independent experiments). F Localization of YAP is demonstrated by immunofluorescence. Scale bar, 25 μm. 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 = 4 independent experiments). *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; n.s.: no significance