ZFIN Image: Zhang et al., 2020, Fig. 1

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Fig. 1 Figure 1. Overexpression of VBP1 inhibits Wnt/β-catenin signaling. A, TOPFlash luciferase assays in HEK293T cells with 500-ng FLAG-tagged VBP1 plasmid transfection. Expression of FLAG-VBP1 was confirmed by Western blotting. Data are from four independent experiments with individual data points shown. B, relative mRNA levels of indicated Wnt target genes in control or VBP1-overexpressing HEK293T cells were analyzed by qRT-PCR. Data are from three independent experiments with individual data points shown. C, TOPFlash luciferase assays in zebrafish embryos with 600-pg VBP1 mRNA injection. Data are from three independent experiments with individual data points shown. D, TOPFlash luciferase assays in Wnt3a-, BIO-, β-CatΔN-, or VP16-Tcf7l1ΔN-treated HEK293T cells with VBP1 overexpression. Control or VBP1-overexpressing HEK293T cells were cotransfected with Renilla and TOPFlash plasmids. The activation of Wnt/β-catenin signal was induced by BIO (1 µm) treatment or by indicated plasmid DNAs (15 ng of Wnt3a, 30 ng of β-CatΔN, or 50 ng of VP16-Tcf7l1ΔN) transfection. Data are from three independent experiments with individual data points shown. E, TOPFlash luciferase assays in wnt3a- or vp16-tcf7l1ΔN-injected zebrafish embryos with VBP1 overexpression. TOPFlash and Renilla plasmids were co-injected into embryos with gfp or VBP1 mRNA. The activation of Wnt/β-catenin signal was induced by injecting 100 pg of wnt3a mRNA or 50 pg of vp16-tcf7l1ΔN mRNA. Data are from three independent experiments with individual data points shown. F, overexpression of VBP1/vbp1 antagonized wnt3a-, β-catΔN-, or vp16-tcf7l1ΔN-induced dorsalized phenotype in zebrafish embryos. Lateral view of representative injected embryos at 5-somite stage was shown. Scale bar, 200 µm. G–I, quantitation of embryos displaying normal or dorsalized phenotype in F. Data are from three independent experiments, and the embryo number of each group is shown on the top. The values are mean ± S.D. Unpaired t test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, nonsignificant.

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