Extended Data Figure 2. Primary validation of Asns as a driver of invasion and metastasis.

a, Representative images of the lungs of mice that were intravenously injected with Asns-silenced or -expressing 4T1-T cells as described in Fig. 2a. b, Quantification of Matrigel invasion capacity for Asns-silenced and -expressing 4T1-T cells (n = 3 replicates per cell line). c, Quantification of mCherry-positive 4T1-T cells after roughly 50% of cells were infected with mCherry-expressing constructs harbouring shRNAs targeting Renilla luciferase and Asns. Cells were grown during the 24-h period that the Matrigel invasion assay described in Fig. 2b was being performed (n = 3 replicates per cell line). d, Violet cell-labelling intensity of Asns-silenced and -expressing 4T1-T cells, relative to the initial population. Cells were grown during the 24-h period that the Matrigel invasion assay described in Fig. 2b was being performed (n = 3 replicates per cell line). e, Free amino-acid quantification by HPLC for each amino acid in Asns-expressing and -silenced cells. Shown are the log-fold changes for each amino acid (n = 3 replicates per cell line). f, Quantification of mCherry-positive 4T1-T cells after roughly 50% of cells were infected with mCherry-expressing constructs harbouring shRNAs targeting Renilla luciferase and Asns. After infection, cells were grown in medium supplemented with l-asparagine or d-asparagine and mCherry percentages were measured at 48 and 96 h (n = 3 replicates per cell line). g, Quantification of Matrigel invasion for Asns-silenced and -expressing cells when assayed in medium supplemented with and without l-asparagine (n = 3 invasion chambers per cell line).