Fig. 4

DDX24 regulates brain and trunk angiogenesis through the Wnt and VEGF pathways, respectively. (A) GO analysis showing the top fold-change enriched terms in HUVECs upon DDX24 knockdown. (B) Fold change of VEGF pathway genes upon DDX24 deficiency in HUVECs and HCMECs. (C) Western blot analysis of the indicated antibodies in HUVECs transfected with control or DDX24 siRNA. Protein levels were normalized to β-actin. n = 3 independent experiments. (D) qRT-PCR analysis of DDX24 and KDR in HUVECs transfected with siNC or siDDX24. (E) qRT-PCR analysis of ddx24 and kdr/kdrl in WT or ddx24−/− zebrafish. (F) Quantification of the ectopic branch of ISV at 54 hpf following injection with CT MO, ddx24 MO, and ddx24 MO + kdr/kdrl MO. n = 36, 38, 37 embryos. (G) Quantification of CtA number at 36 hpf following injection with CT MO, ddx24 MO, and ddx24 MO + kdr/kdrl MO. n = 23, 24, 26 embryos. (H) RNA immunoprecipitation (RIP) followed by RT-qPCR showing enrichment of KDR mRNA by anti-DDX24 antibody in HUVECs. RPL12 mRNA served as a negative control. (I) Predicted top three binding sites and binding motif between DDX24 and KDR mRNA. (J) The percentage of remaining KDR mRNA was tested after the addition of actinomycin D. (K) Fold change of Wnt pathway genes upon DDX24 deficiency in HCMECs and HUVECs. (L) Wnt signaling activity in HCMECs measured by TOPFlash luciferase reporter assay. (M) Western blot analysis of DDX24 and GPR124 in HCMECs transfected with siNC or siDDX24. Protein levels were normalized to β-actin. (N) qRT-PCR analysis of gpr124/reck and Wnt target genes in the head of 36 hpf WT or ddx24−/− zebrafish. (O) Quantification of CtA number at 36 hpf following injection of CT MO, ddx24 MO, or ddx24 MO combined with WT or ∆ICD gpr124 mRNA. n = 38, 40, 40, 40 embryos. All the data are representative of at least three independent experiments. Data are represented as mean ± SD. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, as assessed by one-way ANOVA (C, D, L, and M), nonparametric Kruskal–Wallis test (F, G, and O), parametric two-tailed Student’s t test (E, H, and N) and two-way ANOVA (J).