Fig. 5: FZD7-DAAM1-RhoA-ROCK1/2 supports tumour initiation and metastasis in vivo.

a Schematic of experiment (left) and tumour weight (right) of ROCKi (H1152 or GSK269962A) pre-treated A375M2 cells 18 days post-subcutaneous injection into NSG mice (n = 5 mice for control and H1152, n = 4 for GSK269962A). b–d Quantification of b melanoma cell shape score, c H-score of p-MLC2 staining and d ki-67 positive cells in A375M2 tumours from (a). e Limiting dilution assay estimating TIF (top) and tumour volume (bottom) of shControl and shFZD7 WM1361 cells when injected at different dilutions (500,000, 50,000 and 5,000 cells) into NSG mice (Number of tumours per condition indicated in table). TIF was determined using ELDA. f–h Representative images (top) and quantification (bottom) of f melanoma cell shape score, g H-score of p-MLC2 staining and h ki-67 positive cells in TB and IF of shControl (n = 16) and shFZD7 (n = 9) derived tumours from 50,000 cells’ condition from (e). Scale bar, 100 μm; inset, 25 μm. i Limiting dilution assay estimating TIF (top) and tumour volume (bottom) of shControl and shDAAM1 WM1361 cells when injected at different dilutions (500,000, 50,000 and 5,000 cells) into NSG mice (Number of tumours per condition indicated in table). TIF was determined using ELDA. j–l Representative images (top) and quantification (bottom) of j melanoma cell shape score, k H-score of p-MLC2 staining and l ki-67 positive cells in TB and IF of shControl (n = 10) and shDAAM1 (n = 5) derived tumours from 50,000 cells’ condition from (i). Scale bar, 100 μm; inset, 25 μm. m Representative confocal images (left) and percentage of fluorescence area (right) of mouse lungs 24 h (n = 6 mice) and 3 weeks (n = 5 mice) after tail vein injection into NSG mice of WM1361 cells expressing shControl and shDAAM1. Scale bar, 50 μm. a, m Graphs show mean ± s.e.m. b–l Box limits show 25th and 75th percentiles, the horizontal line shows the median, and whiskers show minimum and maximum range of values. a–d One-way ANOVA with Dunnett post-hoc test. e, i Two-tailed Mann–Whitney test. f–h, j–l One-way ANOVA with Tukey post-hoc test. m Two-tailed t-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. Schematics in this figure were created using Servier Medical Art templates licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com).