Generation of Knockout Cell Lines

SLC7A11-KO, LAMP2 KO, 4EBP1/2 DKO, and RagA/B DKO cell lines were generated using CRISPR/Cas9 technology as previously described^{58 (link)}. In brief, the sgRNAs were cloned into the lentiviral lentiCRISPR v2 vector. All constructs were confirmed by DNA sequencing. pGIPZ-shRNAs against GPX4 were obtained from the Functional Genomics Core at The University of Texas MD Anderson Cancer Center. The sequences of the primers used in PCR mutagenesis, gRNAs, and shRNAs used in this study are listed in Supplementary Table 3. pLenti-SLC7A11-V5 construct was generated as described in our previous publications^{24 (link),59 (link)}. pcDNA-flag-GPX4 expression plasmid was a gift from Dr. Aikseng Ooi at The University of Arizona Health Sciences^{60 (link)}. pCW57.1-4EBP1 4 A construct was obtained from Addgene (#38240). Other reagents were purchased as follows: Erastin (E7781), ferrostatin-1 (SML0583), deferoxamine mesylate salt (DFO, D9533), GSHEE (G1404), MG132 (M7449), chloroquine diphosphate (C6628), and doxycycline (D9891) were obtained from Sigma. Torin1 was obtained from ApexBio (A8312). AZD8055 (S1555) and Salubrinal (S2923) were obtained from Selleckchem. IKE was obtained from MedchemExpress (HY-114481).