a, Dose-dependent toxicity of phenylarsine oxide, indomethacin, auranofin, ivermectin, sunitinib, obatoclax, mitoxantrone, irinotecan, vinblastine, ABT-263, nocodazole, etoposide, paclitaxel, H 2 O 2 and tert-butyl hydroperoxide (tBOOH) of Pfa1 cells expressing mock or FSP1–HA. Cell viability was assessed 24 h after treatment using Aquabluer. b, Dose-dependent toxicity of TNF and staurosporine of mock and FSP1–HA-expressing Pfa1 cells. Cell viability was assessed 24 h after treatment using Aquabluer. c, Immunoblot analysis (ACSL4, HA, cleaved caspase 3 (clv. Casp3), GPX4 and β-actin) of Pfa1 FSP1–HA cells treated with or without TNF for 6 h. d, Immunoblot analysis of FSP1, ACSL4, p53, p21 and VCP expression in p53 (also known as TP53) wild-type and p53-knockout (CRISPR–CAS9-modified) HT1080 cell lines treated with the MDM2 (MDM2 proto-oncogene) inhibitor Nutlin3 or the cytostatic compound doxorubicin (Doxo). Expression of FSP1 was not altered by Nutlin3 or doxorubicin treatment, whereas the expression of p53 and p21 was strongly induced in HT1080 p53 wild-type cells. Data show one representative of n = 3 independent experiments. e, Flow cytometry analysis of annexin V/propidium iodide staining in Pfa1 cells expressing mock or FSP1–HA treated with or without TNF for 4 h. No difference in the apoptotic activity was observed in cells as visualized in the Alexa Fluor 488/PE–Cy5 channels. Data show one representative experiment of an experiment performed independently twice. f, Immunoblot analysis of AIFM1, ACSL4, GPX4 and β-actin in two different Pfa1 Aifm1-knockout cell clones overexpressing mock or AIFM1. Data show one representative of n = 3 independent experiments. g, Dose-dependent toxicity of RSL3, erastin and BSO in Aifm1-knockout Pfa1 cell clones (1 and 2) overexpressing mock or AIFM1. AIFM1 expression does not affect ferroptosis sensitivity. Data are the mean of n = 3 replicates of a representative experiment performed independently three times. h, Time-dependent lactate dehydrogenase (LDH) release of Pfa1 cells stably expressing mock, FSP1–HA or FSP1(G2A) treated with TAM to induce GPX4 loss. Supernatants were collected from 6-well plates at different time points after TAM induction and assayed for lactate dehydrogenase content in a 96-well plate. i, Wild-type and GPX4-knockout HT1080 cells overexpressing mock, hGPX4–FSH, FSP1–HA or FSP1(G2A)–HA treated with and without 200 nM Lip-1. Cell viability was assessed after 72 h using Aquabluer. Data are the mean ± s.d. of n = 3 wells of a 96-well plate from one representative of three independent experiments (a, b, g–i); *P < 0.01; two-way ANOVA. Immunoblot images (c, d, f) are cropped from the chemiluminescence signal files. For gel source data (c, d, f) showing the overlap of colorimetric and chemiluminescence signals, see Supplementary Fig. 1. Source data