Fig. 4: FZD7 downstream of WNT11 supports melanosphere formation and amoeboid invasion via DAAM1.

a, b Heatmap representing log2 fold change in expression of non-canonical Wnt receptors in a A375M2 cells compared to A375P cells or A375M2 cells treated with ROCKi (H1152 and Y27632) and blebbistatin and in b metastatic versus primary melanoma samples from indicated studies from GEO and TCGA databases. c–f After FZD7 or RYK knockdown in A375M2 and WM1361 cells, quantification of c sphere formation index (n = 4 for A375M2, n = 3 for WM1361), d cell viability (n = 5 for A375M2, n = 3 for WM1361), e cell morphology (>300 cells pooled from n = 3) and f p-MLC2 levels (n = 3 for A375M2, n = 4 for WM1361). g Quantification of 3D invasion index through a collagen I matrix after FZD7 knockdown in A375M2 (n = 5) and WM1361 cells (n = 3). h–j After PLCB1 or DAAM1 knockdown in A375M2 and WM1361 cells, quantification of h cell morphology (>250 cells pooled from n = 3), i sphere formation index (n = 4) and j cell viability (n = 6 for A375M2, n = 3 for WM1361). k Quantification of 3D invasion index through a collagen I matrix after DAAM1 knockdown in A375M2 (n = 4) and WM1361 cells (n = 3). l Representative phase-contrast images (left) and quantification of cell morphology (right) of A375P cells on collagen I matrix after WNT11 stimulation and PLCB1 or DAAM1 knockdown (>400 cells pooled from n = 5). Scale bar, 100 μm. m, n Representative immunoblots (top) and quantification (bottom) of m RhoA-GTP in pulldown samples and total RhoA in total lysate (n = 3) and n p-MLC2 levels (n = 4) in A375P cells after WNT11 stimulation and DAAM1 knockdown. c, d, f, g, i–k, m, n Graphs show mean ± s.e.m. e, h, l Box limits show 25th and 75th percentiles, the horizontal line shows the median, and whiskers show minimum and maximum range of values. c–n n means number of independent biological experiments. a, g, k Two-tailed t-test. b Two-tailed t-test with Welch’s correction. c, d, f, i, j One-way ANOVA with Dunnett post-hoc test. e, h Kruskal–Wallis with Dunn’s multiple comparison test. l Kruskal–Wallis test with Benjamini, Krieger and Yekutieli correction. m, n One-way ANOVA with Tukey post-hoc test. For all graphs, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The exact significant p values for *p, **p and ***p are provided in Supplementary Table 1.