Extended Data Fig. 4: WNT5A regulates dormant pathways and slows proliferation.

a, b, Five aged mice per group were intradermally injected with 2.5x10⁵ Yumm1.7 mCherry melanoma cells. After three weeks of tumour growth, mice were treated with 1 mg/kg of a neutralizing sFRP1 or an IgG control antibody via IP injection every 4 days. Representative images are displayed across three independent mice for mCherry positive cells and Ki-67. The number of single cell (less than 10 mCherry positive cells) was assessed at week 5 and quantified per section and presented as mean +/− SEM. Scale bar represents 100 um. A student’s two-way t-test was performed. P = 0.244 c Protein expression analysis was performed on WNT5A low human melanoma samples (FS13, FS14) that underwent lentiviral induced overexpression of WNT5A. HSP90 was used as a loading control. d, e FS13 and FS14 WNT5A overexpressing mCherry human melanoma cells were seeded at a density of 2x10⁴. Cells were then grown over a 10-day period and assessed for proliferation at days 3-5 and 10 using a haemocytometer and further assessed using automated counting via NIS elements. Experiment was performed in biological triplicates with 3 technical replicates each. A two-way ANOVA was performed with a post-hoc Holm-Sidak’s multiple comparisons test for each time point. P-values were taken from day 10. Data were presented as the mean +/- the SEM for each time point. P < 0.0001 for both conditions. f FS13 GFP melanoma cells were seeded at a density 2x10⁴. Cells were treated with 200ng of r-WNT5A every 2 days and assessed for proliferation at days 3-5 and 10 using a haemocytometer and further assessed using automated counting via NIS elements. Experiment was performed in biological triplicates with 3 technical replicates each. A two-way ANOVA was performed with a post-hoc Holm-Sidak’s multiple comparisons test for each time point. P-values were taken from day 10. Data were presented as the mean +/− the SEM for each time point. P < 0.0001 g Western blot analysis was performed on protein lysates from FS13 melanoma cells treated with 200ng of recombinant WNT5A for five days. HSP90 was used as a loading control.