Extended Data Fig. 4: The MAPK-mTORC1-eIF4E Axis is Essential for ATF4 Induction Following Asparagine Limitation.
From: Translational reprogramming marks adaptation to asparagine restriction in cancer
a, Immunoblotting of phosphorylated and total S6 protein in melanoma cells 72 hr after treatment with si-ASNS, si-ATF4, si-BRAF, or indicated combinations. Fig. 3d and Extended Data Fig. 4a show parts of the same experiment and share the internal control (HSP90). b, Immunoblotting of phosphorylated and total S6 protein and 4E-BP1 in melanoma cells 72 hr after treatment with si-ASNS, SCH-772984, or both. c, Immunoblotting of ATF4 and phosphorylated and total S6 protein in Mia-Paca-2 cells 72 hr after treatment with si-ASNS#1 or #2, PD-325901, or a combination of the respective si-ASNS and PD-325901. d, Immunoblotting of ATF4 and phosphorylated and total S6 protein in pancreatic cancer cell lines 72 hr after treatment with si-ASNS, Rapamycin (Rapa), or both. e, Immunoblotting of ATF4 and phosphorylated and total S6 protein in Mia-Paca-2 cells 72 hr after treatment with si-ASNS, Torin 1, or both. f, Proliferation of cancer cell lines measured 72 hr after treatment with si-ASNS, Rapa, or both. Values are shown relative to mock (NT-siRNA and DMSO)-treated cells. Data are representative of three independent experiments and presented as the mean ±SEM of n=3 biological replicates in f. Statistical significance was calculated using two-tailed unpaired Student’s t-test.
