Fig 2: N-sulfo-rich HS clusters are involved in the formation of the Wnt/LRP6 signalosome. a–f The colocalization of phosphorylated LRP6 (pLRP6) and N-sulfo-rich HS clusters. Together with Alexa Fluor 647-dextran (tracer, not shown), mRNAs of wnt8 and lrp6-vsvg were injected into the animal pole region of a ventral blastomere of 4- or 8-cell stage embryos. Signal intensities along white arrows were plotted (b, e), starting (yellow arrowheads) and ending (cyan arrowheads) points as indicated, respectively (a, d). Distributions of pLRP6 and N-acetyl HS were not correlated well (correlation coefficient r = −0.051), whereas distributions of Wnt8 and N-sulfo HS were highly correlated (r = 0.872). Note that pLRP6 staining is colocalized with N-sulfo HS staining on the membrane and also inside cells (white arrowheads) but not with N-acetyl HS staining (open arrowheads). g Requirement of ndst1 for the membrane distribution of endogenous Fzd8. Experimental procedures are essentially the same as in Fig. 6c. Immunostaining was carried out with the anti-Fzd8. std MO, as a negative control, did not affect Fzd8 accumulation at the cell boundary (white arrowheads), whereas ndst1 MO cell-autonomously reduced Fzd8 accumulation at the cell boundary (open arrowheads). Coinjection of ndst1 mRNA recovered Fzd8 accumulation at the cell boundary (white arrowheads). h Requirement of ndst1 for the phosphorylation of LRP6. mRNAs of lrp6, wnt8, and ndst1 (for rescue experiment only, bottom) and indicated MO were injected into the animal pole region of a ventral blastomere of 4- or 8-cell stage embryos. Punctate staining of pLRP6 was impaired by ndst1 MO, which was rescued by ndst1 mRNA. i Quantification of pLRP6 puncta. Graph, mean±s.e.m. Significance level, **p < 0.01 (multiple comparison with pairwise Wilcoxon rank sum test). Numbers of analyzed images (n) are as indicated. See Methods for details. Xenopus embryos (st. 11.5) were fixed, stained, and analyzed. Images are a representative of at least two independent experiments. Amounts of injected mRNA (pg/embryo): lrp6-vsvg, 38; wnt8, 250; ndst1, 15 (h) or 6.7 (g). Amounts of injected MOs (ng/embryo): 28 (g), or 14 (h). Scale bars, 20 μm. a.u., arbitrary units

Fig 3: Emetine suppresses Wnt/β-catenin signaling. (A) Chemical structural formula of emetine. (B-F) SuperTOPFlash reporter gene with empty vector or expression vectors encoding (B) Wnt1; (C) LRP6; (D) Wnt1 and LRP6; (E) dishevelled-2 (DVL2); and (F) β-catenin was used to transfect 293T cells before treatment with vehicle control (DMSO) or emetine (6.25, 12.5, 25, 50 or 100 nM) for 24 h. Luciferase activity was normalized to β-gal activity. 293T cells were transfected with (G) AP-1-Luc reporter along with control vector or a constitutively active Rasv12 expression plasmid, and (H) NFAT-Luc reporter together with control vector or NFATc expression plasmid. Data are the average values from three independent experiments performed in duplicate (*P<0.05). LRP6, low-density lipoprotein-receptor-related protein 6; AP-1, activator protein 1; NFAT, nuclear factor of activated T cells.

Fig 4: VHHs targeting LRP6P3E3P4E4 block cellular responses to Wnt3a. a Schematic representation of LRP6. The P3E3P4E4 module of the extracellular domain was used to generate anti-LRP6 VHHs. Coloring scheme: LRP6P1E1; yellow/orange, LRP6P2E2; pink/orange, LRP6P3E3; blue/orange and LRP6P4E4; green/orange. LA domains are shown in brown. b Wnt luciferase reporter assay performed in LRP6-overexpressing HEK293T cells stimulated with Wnt3a-conditioned medium and treated with 10 μM of the indicated anti-LRP6P3E3P4E4 VHHs. c Wnt luciferase reporter assay performed in HEK293T cells stimulated with Wnt3a-conditioned medium and treated with 10 μM of the indicated anti-LRP6P3E3P4E4 VHHs. Graphs show average (bars) and range (dots) of luciferase activity in duplicate cell cultures transfected in parallel

Fig 5: Emetine inhibits the Wnt/β-catenin signaling pathway in breast cancer cells. (A) MDA-MB-231 and (B) MDA-MB-468 breast cancer cells were treated with emetine at the indicated concentrations for 24 h. (A and B) The expression levels of phosphorylated LRP6, total LRP6, DVL2, active β-catenin and total β-catenin were analyzed by western blotting. A slower-migrating band (upper band) corresponded to phosphorylated DVL2. Western blots were quantitated by densitometry. Each band was presented as corresponding density values normalized to β-actin signal.