Extended Data Fig. 1: ASNS Suppression Induces the Amino Acid Response Pathway.
From: Translational reprogramming marks adaptation to asparagine restriction in cancer
a, GC-MS-based estimation of intracellular asparagine levels in A375 and UACC-903 cells 72 hr after treatment with si-ASNS. b, Relative proliferation of A375 and UACC-903 cells treated with si-ASNS#1 or si-ASNS#2, with or without supplementation with L-asparagine (L-Asn; 0.3 mM), over the indicated time course. c, Immunoblotting of ASNS, ATF4, phosphorylated and total GCN2 and eIF2α in melanoma cells 72 hr after treatment with si-ASNS, L-Asn (0.3 mM), or both. d, Immunoblotting of ATF4 in melanoma lines 72 hr after treatment with si-ASNS, ISRIB, or both (left). Relative proliferation of melanoma cells treated as in (left) for indicated time course (right). Data are representative of three independent experiments and presented as mean ±SEM of n=3 biological replicates in b and d. Data shown as mean ±SEM of n=3 independent experiments in a. Statistical significance was calculated using two-tailed unpaired Student’s t-test for a and two-way Anova for b and d. In b, black and orange P values correspond to the comparison between si-ASNS#1 and si-ASNS#1+L-Asn and si-ASNS#2 and si-ASNS#2+L-Asn respectively.
