Xenbase Image ID: 155690

Figure 1. Uch37 positively regulates Wnt signalling downstream of β-catenin stabilization.(a) TOPflash assay using whole embryos (stage 10.5, 10 embryos were used for each sample). Four-cell stage embryos were animally injected with indicated reagents (150 pg TOPflash reporter; 50 pg pRL-TK; 40 ng Co MO; 40 ng Uch37 MO; 20 pg Wnt8 mRNA; 1 ng Re.Uch37 mRNA). (b) Expression levels of Wnt target genes (siamois and nodal3.1) were examined by RT-PCR analysis using Xenopus animal cap tissues. Two-cell stage embryos were animally injected with indicated reagents (5 pg Wnt8 mRNA; 20 ng Co MO; 20 ng Uch37 MO; 1 ng Re.Uch37 mRNA). Animal cap explants were isolated at stage 9 and cultured until stage 11. WE, Whole embryos; (-), -RT; ODC, ornithine decarboxylase loading control. Full images are presented in Supplementary information (Fig. S12) (c) Axis duplication assay. Four-cell stage embryos were injected in one ventrovegetal blastomere with indicated reagents (5 pg Wnt8 mRNA; 1 ng Uch37 mRNA; 20 ng Uch37 MO). See also Table S2. (d) Quantified result of c. (e) TOPflash assay in HepG2 cells. After transfection of reporter constructs into stable cells (sh Control or sh Uch37) for 48 h, luciferase activity was measured. Knockdown of β-catenin indicates a positive control for downregulation of Wnt activity. (f) Quantitative real-time PCR (qPCR) analysis for the expression of Wnt-target genes (cyclinD1 and c-myc) in stable HepG2 cells. Knockdown of β-catenin indicates a positive control for downregulation of Wnt activity. The quantities of indicated mRNA were normalized by β-actin. Data represent average values from three independent experiments performed. Error bars indicate standard deviations of triplicate. *p < 0.002; **p < 0.001 (two-tailed Student’s ttest). (g) Axis duplication assay. Four-cell stage embryos were injected in one ventrovegetal blastomere with indicated reagents (5 pg Wnt8 mRNA; 200 pg Dvl2 mRNA; 25 pg β-catenin mRNA; 20 ng Co MO; 20 ng Uch37 MO). See also Supplementary Fig. S5a and Table S3). Image published in: Han W et al. (2017) Copyright © 2017, The Author(s). This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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