To more directly assess the role of NANOS1, PUM2, and CPSF4 in metastasis, we knocked down (KD) their expression in two metastatic mouse mammary cancer cell lines, 4T1 and 6DT1, using short hairpin RNAs (shRNAs) (Supplementary  Fig. S1a, b) and performed spontaneous metastasis assays by mammary fat pad injection into syngeneic (BALB/c for 4T1, FVB/N for 6DT1) mice (2). This evidence concerns the gene PUM2 and breast cancer.