Figure 7.
Glucose deprivation activates the GCN2-eIF2α-ATF4 pathway. (A) HT1080 cells were incubated with/without 25 mM glucose for 16 h. Whole cell lysates (WL) were harvested for immunoblot (IB) or immunoprecipitation (IP). (B) HT1080 cells were incubated in glucose-free DMEM for up to 16 h with indicated concentrations of glutamine added and lysates were subjected to immunoblotting. (C) Cells were deprived of Glucose for 2 and 4 h and the levels of individual amino acids were analysed by LC-MS. (D) Wild-type, GCN2−/− and PERK−/− MEFs were incubated in glucose-free DMEM for up to 8 h, cells were harvested for immunoblot. Glutamine deprivation treatment was for 4 h. (E) HT1080 cells were incubated with/without 25 mM glucose for 8 h, total RNA was extracted for real-time PCR for the ATF4 targets ASNS (left) or SLC1A4 (right). (F) HT1080 cells were cultured with/without 25 mM glucose, 2 × 104 cells/well. shATF4 cells were supplemented with 1 × NEAA. After 24 h, cell survival was measured using MTT assay and survival was normalized to that of the high glucose group.