Regulation of ATF4 and Rictor in Cell Lines

The eIF2αP-proficient or -deficient MEFs, HT1080 and A549 tumor cells were generated as described previously.^{30 (link)} ATF4 KO MEFs as well as PERK/GCN2 KO MEFs were previously described.^{41 (link), 59 (link)} Cells were cultured in Dulbecco modified Eagle medium (Wisent, St-Bruno, QC, Canada) supplemented with 10% fetal bovine serum (FBS; Gibco, Burlington, ON, Canada), antibiotics (100 U/ml of penicillin–streptomycin; Gibco) and 2.5 μg/ml of puromycin (Sigma, Oakville, ON, Canada). The shRNA-mediated KO of ATF4 in HT1080 cells was carried out based on previously reported protocol.^{69 (link)} Lentiviral shRNA targeting ATF4 (TRCN0000013573) was obtained from the RNAi Consortium (TRC) arrayed human genome-wide shRNA collection (Sigma). shRNA-mediated KO of Rictor was performed as described previously.^{56 (link)} H₂O₂ was purchased from Bioshop, Canada; GDC-0941 was obtained from Selleckchem, USA; thapsigargin, rapamycin, Akt1,2 inhibitor, PAO, PEITC and erastin were obtained from Sigma.