Extended Data Fig. 1: Identification and characterization of FSP1 as an anti-ferroptotic protein.
From: FSP1 is a glutathione-independent ferroptosis suppressor
a, Schematic depicting the generation of a lentiviral cDNA-overexpression library using the total mRNA from MCF7 cells. b, Genomic PCRs of the 14 Pfa1 cell clones that remained clones after the removal of false-positive results using human-specific primers to amplify the human cDNAs of GPX4 (571 bp) or AIFM2 (524 bp). The clones 2, 16, 24, 25, 26, 28 and 30 showed positive PCR results for GPX4 (571 bp). Clones 1, 44, 45, 50, 51, 52 and 53 were positive for AIFM2 (524 bp) as indicated by the white arrows. Data are one of n = 3 independent experiments. c, Immunoblot analysis of ACSL4, HA, GPX4 and β-actin expression in Pfa1 cells stably expressing mock or FSP1–HA. Gpx4 deletion was induced by the administration of TAM for the indicated time period. d, Immunoblot analysis of ACSL4, HA, GPX4 and β-actin expression in wild-type and GPX4-knockout HT1080 cells stably expressing mock, GPX4–FSH or FSP1–HA. e, Proliferation of mock and FSP1–HA Pfa1 cells treated with or without TAM. Cell numbers were assessed every 24 h using a Neubauer haemocytometer. Data are mean ± s.d. of n = 3 wells of a 24-well plate from one representative of two independent experiments. f, g, Dose-dependent toxicity of erastin (f) and l-buthionine sulfoximine (BSO; g), which is an inhibitor of γ-glutamyl-cysteine ligase, in Pfa1 cells expressing mock or FSP1–HA. h, i, Dose-dependent toxicity of erastin (h) and BSO (i) in HT1080 cells expressing mock or FSP1–HA. Cell viability was assessed 48 h (f, h) or 72 h (g, i) after treatments using Aquabluer. Data are mean ± s.d. of n = 3 wells of a 96-well plate from one representative of three (f–i) independent experiments. *P < 0.01; two-way ANOVA. j, Measurement of total glutathione levels in Pfa1 mock, FSP1-expressing and FSP1-expressing Gpx4-knockout cells treated with or without BSO. Data are mean ± s.d. of n = 3 wells of a 96-well plate from one representative of three independent experiments. k, Left, determination of NADPH consumption by glutathione reductase as an indirect measure of the GPX4 activity. Phosphatidylcholine lipid hydroperoxide (PCOOH) was used as a GPX4-specific substrate. Right, cell lysates from mock and FSP1–HA Pfa1 cells treated with or without TAM for 48 h were used for the assay as shown by the immunoblot (FSP1, GPX4 and β-actin). FSP1 was detected using the polyclonal antibody (PA5-24562). Data are mean ± s.d. of n = 3 wells of a 6-well plate from one representative of three independent experiments. Immunoblot images (c, d, k) are cropped from the chemiluminescence signal files. For gel source data (c, d, k) showing the overlap of colorimetric and chemiluminescence signals, see Supplementary Fig. 1.
