Identification and validation of tryptophan metabolites as potent ferroptosis inhibitor. A) Schematic of the identification of potential ferroptosis‐resistant amino acid‐derived metabolites, using HT1080 cells pretreated with indicated metabolites followed by RSL3 treatment for 24 h. B) Heatmap data showing the change of ferroptotic cell death upon amino acid metabolites treatment in HT1080 cells. C) Schematic diagram of tryptophan metabolism pathway. Trp, L‐tryptophan; N‐FKYN, N‐formyl‐kynurenine; L‐KYN, L‐kynurenine; 3‐HK, 3‐hydroxykynurenine; 3‐HA, 3‐hydroxyanthranillic acid; 5‐HTP, 5‐hydroxytryptophan; 5‐HT, serotonin; 5‐HIAA, 5‐hydroxyindoleacetic acid. D) Dose‐dependent toxicity of RSL3 induced cell death in a panel of cancer cell lines with or without 5‐HT treatment. Cell viability was assessed 24 h thereafter using CCK8. E) Representative phase‐contrast images of HT1080 cells treated with 5‐HT (10 µm), RSL3 (200 nm), and Fer‐1 (1 µm) for 8 h. Dead cells were stained with Sytox Green. Scale bars, 50 µm. F) Cell death measurement of HT1080 cells treated with 5‐HT (10 µm), RSL3 (200 nm), and Fer‐1 (1 µm) for 8 h. G) Lipid peroxidation measurements in HT1080 cells treated with 5‐HT (10 µm), RSL3 (200 nm), and Fer‐1 (1 µm) for 4 h. H) Cell death measurement of HT1080 GPX4‐/‐ cells treated with tryptophan (10 µm), 5‐HTP (10 µm), 5‐HT (10 µm), 5‐HIAA (10 µm), RSL3 (200 nm), and Fer‐1 (1 µm) for 8 h.