Generating and Maintaining Cell Lines

Primary as well as immortalized eIF2αP-proficient or deficient MEFs, IMR90, BJ-hTERT, HT1080, and A549 cells were generated and maintained as described previously [18 (link), 19 (link)]. ATF4 KO MEFs as well as HT1080 cells expressing ATF4 shATF4 were established as previously described [18 (link)]. LATS1 KO MEFs were previously reported [52 (link)]. The shRNA-mediated knockdown of LATS1 in HT1080 cells was performed with as previously described targeting vector [53 (link)]. Cells were cultured in Dulbecco modified Eagle medium (DMEM; Wisent) supplemented with 10% fetal bovine serum (FBS; Gibco), antibiotics (100 U/ml of penicillin-streptomycin; Gibco) and 2.5 μg/ml of puromycin (Sigma). H₂O₂ was purchased from Bioshop, Canada; thapsigargin, trolox, phenylarsine oxide (PAO) and propidium iodide were obtained from Sigma; MG132 was purchased from Enzo Life Sciences. In control treatments, an equivalent volume of the solvent of each drug was added in culture.