Fig. 2

Figure 2. VBP1 is required for Wnt/β-catenin signaling activity. A, VBP1 protein levels in stable shRNA-treated HEK293T and U2OS cells. Densitometric analysis was from three independent experiments with individual data points shown. B and C, the transcriptional levels of indicated Wnt target genes in control or VBP1-depleted HEK293T (B) and U2OS (C) cells were analyzed by qRT-PCR. Data are from three independent experiments with individual data points shown. D, representative images of injected embryos. GFP fluorescence was detected at the midbrain in TOPdGFP transgenic embryos at 24 hpf. The pattern of otx2 expression did not change in the corresponding injected embryos. Scale bar, 200 µm. E, quantification of green fluorescence in (D) as relative fluorescence intensity. Image J software was used to measure the fluorescence intensity. Data are from three independent experiments, and the embryo number of each group is shown on the top. F, Wnt/β-catenin activity in Wnt3a-, LiCl-, β-CatΔN-, and VP16-Tcf7l1ΔN-treated cells with VBP1 knockdown. TOPFlash plasmid was cotransfected with Renilla plasmid into control or VBP1-knockdown cells. Wnt activity was induced by LiCl (30 mm) or indicated plasmid DNAs (10 ng of Wnt3a, 15 ng of β-CatΔN, or 30 ng of VP16-Tcf7l1ΔN). Data are from three independent experiments with individual data points shown. G, TOPFlash luciferase assays in shRNA-expressed U2OS (left) or HEK293T cells (right). Wnt/β-catenin activity was rescued by a shRNA-resistant zebrafish Vbp1. The activation of Wnt/β-catenin signal was induced by LiCl treatment (left) or transfection with β-CatΔN (right) plasmid DNA. Data are from three independent experiments with individual data points shown. The values are mean ± S.D. Unpaired t test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. One-way analysis of variance followed by Tukey's post-test. Groups labeled with different letters are significantly different from each other.