Figure 1. Schematic of the B16-F10 pulmonary metastasis screen.

B16-F10 cells are passaged 2 days prior to intravenous administration to ensure they are in log-phase growth when collected. Cells are collected and prepared for administration to wildtype control and mutant mice that are culled 10 days later for counting B16-F10 colonies on the lungs. The total count is determined and for each mutant line (MT1 and MT2) the metastatic ratio is derived by comparing the average of the concurrent wildtype controls and the average of the mutant cohort (see example values given) and significance determined by Mann-Whitney test. Additional cohorts are screened when the Mann-Whitney P value was≤0.0175 and the metastatic ratio was≤0.6 or≥1.6, and all the data combined to run through an Integrative Data Analysis.