Extended Data Fig. 6: Role of MER and AXL in metastatic dormancy and reactivation.

a, b, Analysis of melanoma cell lines from the Cancer Cell line Encyclopedia (CCLE) dataset were performed on human samples stratified into the top (AXL high) and bottom (AXL low) 50^th percentile of AXL expression (N = 30) and presented as a heat map of the average Z-score. An unpaired two-way t-test was performed on each condition, investigating previously established dormancy and proliferative genes, with P < 0.05 considered significant. c, d Analysis of MER high and MER low cells from the CCLE as described above. e TCGA analysis of relative mRNA expression of MER in patient tumour samples comparing primary vs metastatic (distant metastases only) tissue sites and presented as a violin plot, with quartiles represented by black lines and the median by the broken line (N = 104, 68). A student’s two-way t-test was performed. P = 0.0023. f 1205Lu shAXL and control human mCherry 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 = 0.0160 (AXL SH1) and P = 0.340 (AXL SH2) when compared with the negative control group. g Protein expression analysis was performed on protein lysates of lentiviral induce shRNA knockdown of MER in FS14 human melanoma cells. HSP90 was used as a loading control. h, i FS13 and FS14 shMER and control human mCherry 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 triplicate 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 all groups compared with the negative controls. j Protein expression analysis was performed on protein lysates of Yumm1.7 Dox inducible MER overexpressing mCherry mouse melanoma cells treated with Dox (0.5ug/ml) vs a No Dox control for 48 h. HSP90 was used as a loading control. k Yumm1.7 Dox inducible MER overexpressing mCherry mouse 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 triplicate 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. l TCGA analysis of relative mRNA expression of MER in patient samples stratified into stage I/II vs. Stage III/IV and presented as a violin plot, with quartiles represented by black lines and the median by the broken line (N = 219, 197). A student’s two-way t-test was performed. P = 0.0025. m Tumour volumes from young mice (N = 9 for Dox Day 21, N = 8 for Dox Day 3, N = 7 for No Dox due to mouse deaths) subdermally injected with 2.5x10⁵ Yumm1.7 Dox inducible MER mCherry melanoma cells. Mice were treated with Dox (2 mg/ml in water) from day 3 or day 21 onwards, with tumours measured for 5 weeks. 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 31. Data were presented as the mean +/− the SEM for each time point. *** P = 0.0004 comparing No Dox with Dox Day 3. * P = 0.0281 comparing No Dox with Dox Day 21. n Western blot analysis was performed on protein lysate from Yumm1.7 Dox-inducible AXL overexpressing mCherry melanoma cells comparing control and Dox (0.5ug/ml) treated cells over 48 h. HSP90 was used as a loading control. o Yumm1.7 Dox inducible AXL overexpressing mCherry mouse 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 = 0.0272. p Tumour volumes from aged mice (N = 8 for No Dox, N = 5 for Dox Day 3 and N = 7 for Dox Day 21 due to mouse deaths) per group were subdermally injected with 2.5x10⁵ Yumm1.7 Dox inducible AXL mCherry melanoma cells. Mice were treated with Dox (2 mg/ml in water) from day 3 or day 21 onwards, with tumours measured for 5 weeks. 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 36. **** P < 0.0001 for comparisons between No Dox with both Dox Day 3 and Dox Day 21. Data were presented as the mean +/- the SEM for each time point.