Extended Data Fig. 4: FSP1 protects against unrestrained lipid peroxidation in a COQ2-dependent manner.
From: FSP1 is a glutathione-independent ferroptosis suppressor
a, Enhanced resolution confocal microscopy images demonstrating different localizations of FSP1–GFP and the FSP1(G2A)–GFP mutant in HT1080 cells. DAPI (yellow), GFP (green), endoplasmic reticulum or Golgi tracker (magenta). Scale bars, 20 nm. Data show one representative of n = 3 independently performed experiments. b, Formation of 5-hydro(pero)xyeicosatetraenoic acid (5-H(P)ETE) (multiple reaction monitoring (MRM): 319 → 115), 12-H(P)ETE (MRM: 319 → 179) and 15-H(P)ETE (MRM: 319 → 219) in either mock (black) or FSP1–HA-overexpressing (red) Pfa1 cells treated with 0.2 μM RSL3 and 40 μM arachidonic acid. Hydroperoxides were analysed as their alcohols following reduction with PPh3 (triphenylphosphane) in methanol. Data are the mean of biological triplicates from one representative of n = 3 independently performed experiments. c, Dose-dependent rescue of three independent COQ2-knockout HT1080 cell clones (56, 61 and 68) by supplementation of the cell culture medium with uridine, CoQ10 or decyl-ubiquinone. Cell viability was assed using the Aquabluer assay 48 h after treatment. Data are mean ± s.d. of n = 3 wells of a 96-well plate performed once. d, Immunoblot analysis of FSP1 and β-actin in HT1080 parental (left) and HT1080 COQ2-knockout (56) (right) cells overexpressing FSP1–GFP, FSP1(G2A)–GFP or GFP. Immunoblot images are cropped from the chemiluminescence signal files. For gel source data showing the uncropped chemiluminescence signals, see Supplementary Fig. 1. e, SDS gels showing the different purification steps of recombinant FSP1 from bacterial cell lysates. Left, SDS gel of protein extracts after initial nickel affinity chromatography (E1), the SUMO-tag was cleaved in the eluate by addition of the SUMO protease (dtUD1) and a second round of nickel affinity chromatography was performed to remove the cleaved SUMO-tag as well as uncleaved SUMO–FSP1 and SUMO protease (E2). The flow-through fraction was collected (second nickel). The SUMO–FSP1 fusion protein is visible around 55 kDa and FSP1 at 40.5 kDa. Right, SDS gel showing different fractions containing FSP1 40.5 kDa (A8–A12, B1–B7 and C3–C4) from size-exclusion chromatography of FSP1 after the second nickel-affinity chromatography. Fractions C3 and C4 were used for subsequent assays. One representative of at least three independent experiments.
