1. Non-acylated Wnts Can Promote Signaling
Kelsey F. Speer, Anselm Sommer, Benjamin Tajer, Mary C. Mullins, Peter S. Klein, Mark A. Lemmon 
Cell Reports 
Volume 26, Issue 4, Pages e5 (January 2019)
DOI: /j.celrep
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2. Cell Reports 2019 26, 875-883.e5DOI: (10.1016/j.celrep.2018.12.104)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1 Effects of Site 1 and Site 2 Mutations on xWnt8 Activity
(A) Crystal structure of the xWnt8/mFZD8 CRD complex (PDB: 4F0A), with “thumb” and “index finger” projections on xWnt8 binding to the CRD at sites 1 and 2, respectively (Janda et al., 2012). Residues mutated in site 1 (green) and site 2 (blue) are marked. The palmitoleoyl chain and S187 are red.
(B) Dorsalization phenotypes observed upon ectopic xWnt8 expression in ventral cells of X. laevis embryos. The top panel shows tailbud-stage embryos with archetypal Wnt overexpression phenotypes and corresponding scores. Representative xWnt8 mutant phenotypes are shown in the bottom two rows. Yellow arrow, partial axis duplication; black, full axis duplication; red, radial dorsalization.
(C) Quantitation of dorsalization phenotypes in Xenopus embryos for site 1 and site 2 mutations. Total number of embryos scored (across three biological replicates) is listed for each bar. Dorsal scores for xWnt8WT and xWnt8S187A are from the dataset in Figure 2A, represented here for comparison.
(D) Initial RT-PCR quantitation of Siamois and Xnr3 induction for each variant, represented as mean ± SEM (n = 3). Significance is denoted as ns (p ≥ 0.05), ∗ (p ≤ 0.05), ∗∗ (p ≤ 0.01), or ∗∗∗ (p ≤ 0.001), for two-sided p values.
(E) Expression of injected xWnt8 variants assessed by western blotting of mid-gastrula-stage embryos. Representative of at least three repeats.
(F) Dorsalization phenotypes observed in zebrafish embryos upon ectopic expression of xWnt8WT or xWnt8S187A mRNA. Pictures (top row) show representative embryos at 1 day post fertilization displaying normal (left), moderately dorsalized (“twisted,” center), or highly dorsalized (“bustled,” right) phenotypes. Quantitation of observed phenotypes is shown below, with number of embryos scored across at least two biological replicates listed for each bar.
See also Figure S1.
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4. Figure 2 xWnt8 Retains Biological Activity without Acylation
(A) xWnt8S187A causes dorsalization of Xenopus embryos. Data are represented both as mean dorsal score (left, with number of embryos injected above each bar) and phenotype frequency (right), scored across four biological replicates.
(B) Dose-response curve for Siamois and Xnr3 expression (RT-PCR) induced by xWnt8WT and xWnt8S187A (mean ±SEM, n = 3), as in Figure 1D. Although the difference in maximal Xnr3 expression at 500 pg mRNA for xWnt8WT and xWnt8S187A appears statistically significant (p ≤ 0.05), its biological significance is unclear.
(C) Inhibition of xWnt8WT and xWnt8S187A signaling by xFZD8 CRD. xWnt8 and xFZD8 CRD mRNA were co-injected and RT-PCR performed at the gastrula stage. Siamois and Xnr3 induction is represented as in Figure 1D (mean ± SEM, n = 4).
(D) xWnt8WT and xWnt8S187A proteins co-immunoprecipitate with myc-tagged xFZD8 CRD in embryos co-injected with both mRNAs. The band in lanes 2 and 6 of the α-myc immunoprecipitation (IP) blot corresponds to mouse α-myc antibody heavy chain, which co-migrates with the CRD (∼50 kD). Representative of at least three biological repeats. Significance is denoted as ns (p ≥ 0.05), ∗ (p ≤ 0.05), ∗∗ (p ≤ 0.01), or ∗∗∗ (p ≤ 0.001), for two-sided p values.
See also Figure S2.
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5. Figure 3 mWnt1 Requires Acylation for Biological Activity
(A) mWnt1S224A does not dorsalize Xenopus embryos. Mean dorsal scores (left) were calculated for tailbud-stage embryos, with injected embryo number listed above each bar (scored across 3 biological replicates). Right: photographs of representative embryos.
(B) mWnt1S224A fails to induce Siamois or Xnr3. Data presented as in Figure 1D (mean ± SEM, n = 3), with significance denoted as ns (p ≥ 0.05), ∗ (p ≤ 0.05), ∗∗ (p ≤ 0.01), or ∗∗∗ (p ≤ 0.001), for two-sided p values.
(C) mWnt1WT and mWnt1S224A proteins are expressed at similar levels as assessed by western blotting of injected mid gastrula stage embryos. Representative of at least two biological repeats.
See also Figure S3.
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6. Figure 4 Effects of Acylation Site Mutations in hWnt3a and mWnt5a
(A) hWnt3aS209A dorsalizes Xenopus embryos. Mean dorsal score data (left) are represented as in Figure 2A, with injected embryo number listed above each bar (scored across three biological replicates, with representative embryos shown at right (arrows as in Figure 1B).
(B) Signaling by hWnt3aWT and hWnt3aS209A is inhibited by xFZD8 CRD (mean ± SEM, n = 3), as described for xWnt8 in Figure 2C, with significance denoted as ns (p ≥ 0.05), ∗ (p ≤ 0.05), ∗∗ (p ≤ 0.01), or ∗∗∗ (p ≤ 0.001), for two-sided p values.
(C) Co-immunoprecipitation, as in Figure 2D, shows that hWnt3aWT and hWnt3aS209A both interact with xFZD8 CRD. The faint band in lanes 2 and 6 of the α-myc IP blot is α-myc heavy chain. Representative of at least three biological repeats.
(D) mWnt5aS244A does not affect CE in Xenopus embryos. Data represent the percent of individuals injected with mWnt5aWT or mWnt5aS244A mRNA displaying CE defects at neurula stages, with the number of scored individuals (across 3 biological replicates) listed above each bar. Photographs show representative tailbud stage embryos injected with 0 pg and 100 pg mRNA.
(E) mWnt5aWT and mWnt5aS244A proteins are expressed at similar levels in mid-gastrula-stage embryos.
See also Figure S4.
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