Cell lines
B16-F0 (ATCC; CRL-6322) and its LN metastatic derivatives: NBF0-LN1-18IL, NBF0-LN7-1112AR, NBF0-LN7-1120BL, NBF0-LN7-1134BL, NBF0-LN8-1194BR, NBF0-LN8-1198AR, NBF0-LN8-1205BL, NBF0-LN9-1315BL and NBF0-LN9-1358IR—were provided by the Reticker-Flynn Laboratory. For simplicity, these cell lines are referred to throughout the manuscript as: B16-F0, LN1-18IL, LN7-1112AR, LN7-1120BL, LN7-1134BL, LN8-1194BR, LN8-1198AR, LN8-1205BL, LN9-1315BL and LN9-1358IR, respectively. B16F10 wild-type (WT), B16F10 Fsp1-KO and B16F10 Gpx4-KO cells were obtained from the Conrad Laboratory. B16-F0 Fsp1-KO, LN7-1134BL Fsp1-KO, LN9-1315BL Fsp1-KO, B16-F0 Gclc-overexpression, LN7-1134BL Gclc-overexpression, B16-F0 Gclc-KO and B16-F0 Nrf2-overexpression lines were generated in this study. Human melanoma cell lines MeWo, SK-MEL-5, A375, murine melanoma lines Yale University Melanoma Model (YUMM) 3.3 and YUMM 5.2, and HEK293T cells were purchased from ATCC. All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, 11885076) supplemented with 10% FBS (Thermo Fisher Scientific, 26400044) and 1% penicillin–streptomycin (Thermo Fisher Scientific, 15140122). All of the other lines were authenticated by ATCC using STR profiling. Cells were routinely tested for mycoplasma contamination using MycoStrip (InvivoGen, rep-mys-50).
Chemicals
RSL3 (HY-100218A), erastin-2 (HY-139087), iFSP1 (HY-136057), BTZ (HY-10227) and PEG300 (HY-Y0873) were purchased from MedChemExpress. ML-210 (S0788), MG-132 (S2619) and icFSP1 (E1535) were acquired from Selleck Chemicals. Rotenone (R8875), oligomycin (75351), antimycin A (A8674), L-BSO (B2515), N-acetyl cysteine (A9165), Na 2 SeO 3 (S5261), CQ (C6628) and PEG400 (202398) were obtained from Sigma-Aldrich. FCCP (15218), MTT (21795), GSHee (14953), liproxstatin-1 (17730), IMP-1088 (25366), NSC 624206 (20569), FSEN1 (38025), viFSP1 (39927) and triacsin C (10007448) were obtained from Cayman Chemical Company. MitoView Fix 640 (70082) and LipidSpot 488 (70065) were sourced from Biotium. Lipofectamine 3000 (L3000015), Bodipy 581/591 C11 (D3861), SYTOX Green (S7020), Lysotracker Deep Red (L12492) and NucBlue Live ReadyProbes Reagent (R37605) were from Thermo Fisher Scientific.
Plasmids
pCMV3-FSP1-OFP plasmid (MG52065-ACR) was obtained from Sino Biological. Lenti-luciferase-P2A-neo (Addgene, 105621), psPAX2 (Addgene, 12260), pMD2.G (Addgene, 12259) and PX458 (Addgene, 48138) were obtained from Addgene. Custom constructs including pTWIST-mFSP1-G2A-OFP, pLVX-EF1α-GCLC-IRES-Hygro, and pLVX-EF1α-NRF2-IRES-Hygro were synthesized by Twist Bioscience and cloned into expression vectors using Gibson Assembly.
Generation of stable cell lines
Stable cell lines expressing luciferase, GCLC or NRF2 were generated through lentiviral transduction followed by antibiotic selection. Lentivirus was produced by co-transfecting HEK293T cells with 5 µg of either Lenti-luciferase-P2A-neo, pLVX-EF1α-GCLC-IRES-Hygro or pLVX-EF1α-NRF2-IRES-Hygro, combined with 5 µg psPAX2 and 0.5 µg pMD2.G using Lipofectamine 3000. Virus-containing supernatants were collected every 24 h for 48 h, filtered and supplemented with 8 µg ml−1 Polybrene (Sigma-Aldrich, H9268). Target cells were infected and subsequently selected with either 1,500 µg ml−1 G418 or 1,000 µg ml−1 hygromycin B for 6 days to establish stable populations.
CRISPR–Cas9-mediated gene KO
To generate Fsp1- or Gclc-KO cell lines in B16-F0 and its LN metastatic derivatives, sgRNAs were designed with BbsI-compatible overhangs and cloned into the PX458 Cas9-GFP vector. The sgRNA sequences were as follows: Fsp1 (CACCGGCGGCTGCCAGCCAGCTGC) and Gclc (CACCGGGGAGTTACATGATCGA). sgRNA insertion was confirmed by whole-plasmid sequencing. Cells were transfected with PX458-sgRNA constructs using Lipofectamine 3000 and GFP-positive cells were sorted by flow cytometry and expanded. Transfection and cell sorting was repeated a second time to generate a pure population for expansion prior to validation. KOs were validated by western blotting and Sanger sequencing (Extended Data Fig. 8i,j for FSP1 and Extended Data Fig. 5j for GCLC).
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