Extended Data Fig. 10: PROS1 promotes melanoma growth.

a–e, 1205Lu (P < 0.0001), FS4 (P < 0.0054), FS13 (P = 0.0015), FS14 (P < 0.0001) and Yumm1.7 (P < 0.0001). fluorescent melanoma cells were treated every two days with 500ng of r-PROS1 every 2 days vs a PBS control 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 and P < 0.05 was designated significant. Data were presented as the mean +/− the SEM for each time point. f FS14 shMER knockdown cells and control cells were seeded at a density of 2x10⁴. Cells were then grown over a 10-day period, while being treated with 500 ng of r-PROS1 every two 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 for control + PROS1 vs all other treatment groups. g Protein lysate from Yumm1.7 cells treated with 500 ng of r-PROS1 for 48 h underwent western blot analysis. HSP90 was used as a loading control. h, i Young mice were intradermally injected with 2.5x10⁵ Yumm1.7 mCherry melanoma cells (5 mice per group). After three weeks of tumour growth, mice were subsequently treated with 500 ng of rPROS1 or PBS via IP injection every 2 days. After 5 total weeks, Lungs were taken, PFA embedded and underwent IHC analysis of mCherry positive melanoma cells. Both groups were further assessed for the average number of single cell colonies (<10 mCherry positive cells) per high powered frame (20 x) across entire lung sections and presented as mean +/− SEM. A student’s two-way t-test was performed. Representative images are displayed across three independent mice for mCherry positive cells and Ki-67 with the scale bar representing 100 um. P = 0.8757. j 5 young mice per group were intradermally injected with 2.5x10⁵ Yumm1.7 mCherry melanoma cells. After three weeks of tumour growth, mice were subsequently treated with 500 ng of r-PROS1, r-SFRP1, a combination of both or PBS via IP injection every 2 days. After 5 total weeks, lungs were taken, PFA embedded and subjected to IHC analysis of mCherry positive melanoma cells. Representative images were displayed for mCherry and Ki-67 with the scale bar representing 100 um.