GoKawiila, Fluorescence images of CRISPR Ctrl, MFF KO or MFF-rescued HeLa cells, with or without ρ0 induction, stained with TMRE and Hoechst. Scale bars, 10 µm. b, Immunoblotting of MFF and PEX5 in indicated MFF backgrounds, with or without PEX5 knockout. c, Immunoblotting and cell death in untreated HeLa cells or ρ0 cells with siRNA-mediated PEX5 knockdown after treatment with vehicle or 150 nM RSL3 for 24 h, with or without Lip-1 pretreatment. d–f, ATP levels (d), OCR responses (e) and ETC complex I–V activities (f) in CRISPR Ctrl or MFF KO cells after RSL3 treatment; cells were treated with 200 nM RSL3 for 6 h in d and f, with or without Lip-1 pretreatment, or 1 µM RSL3 for 24 h in e. g, Streptavidin blotting of PMP70 and TOM20 in miniTurbo–TOM20–HA-expressing HeLa cells after 200 nM RSL3 for the indicated times. h, Peroxisomal ROS in untreated or ρ0 cells and mitochondrial ROS in wild-type or PEX5-deficient cells after 200 nM RSL3 for 60 min, with or without Lip-1 pretreatment. i, Cytosolic mtDNA levels in wild-type or MFF KO HeLa cells after 200 nM RSL3 addition at 0 min. The red arrow indicates the time point showing a significant difference. j, Cytosolic mtDNA levels in MFF KO HeLa cells with stable MFN1 or MFN2 knockout after 200 nM RSL3 for 6 h, with or without 0.5-h pretreatment with 2 µM Lip-1. k, Cytosolic mtDNA levels in HeLa cells treated with 200 nM RSL3 for 6 h and cell death in HeLa cells treated with 150 nM RSL3 for 24 h after 0.5-h pretreatment with the indicated pharmacological inhibitors. l, Cytosolic mtDNA levels in HeLa cells treated with or without 200 nM RSL3 for 6 h; the inset shows mtDNA primer positions and the dashed arrow indicates mtDNA release. m, VDAC1 oligomerization in CRISPR Ctrl, MFF KO, and DRP1 KO HeLa cells treated with vehicle or 200 nM RSL3 for 6 h, with Lip-1 pretreatment where indicated, followed by EGS crosslinking. n,o, VDAC1 oligomerization (n) and cytosolic mtDNA levels (o) in HeLa cells treated with 200 nM RSL3, 10 µM Mito-TEMPO or both for 6 h. p, Immunoblotting of cGAS–STING pathway activation in CRISPR Ctrl or MFF KO cells treated with or without 200 nM RSL3 for 6 h. q, Immunoblotting and cell death in HeLa cells expressing sgNC or sgRNAs targeting cGAS or STING after 150 nM RSL3 for 24 h, with or without Lip-1 pretreatment. r, Hierarchical clustering of eicosanoid metabolomics data from CRISPR Ctrl or MFF KO cells after 200 nM RSL3 for 6 h. s, Mitochondrial and peroxisomal ROS and cytosolic mtDNA levels in CRISPR Ctrl or MFF KO HeLa cells preincubated with vehicle or 2 µM 17-HETE for 4 h, followed by vehicle or 150 nM RSL3 for 6 h. t, PCA of untargeted metabolomics data from HeLa cells treated with vehicle, 17-HETE, RSL3 or their combination. u, Immunoblotting of CYP4A11, CYP4F2, CYP4F3 and CYP4F11 in HeLa cells treated with vehicle, 25 µM erastin for 24 h or 200 nM RSL3 for 6 h. v, Immunoblotting and cell death in HeLa cells expressing sgNC or CYP4A11-targeting sgRNAs after vehicle or 150 nM RSL3 for 24 h, with or without Lip-1 pretreatment. w, Cell death in CRISPR Ctrl or MFF KO cells with or without CYP4A11 overexpression after 100 nM RSL3 for 24 h, with or without Lip-1 pretreatment. x, Representative images of mitochondrial morphology in CRISPR Ctrl and MFF-KO cells expressing CYP4A11-targeting sgRNAs after treatment with vehicle, 200 nM RSL3 or 2 µM 17-HETE for 4 h. Scale bars, 5 µm. y, Cell death in HeLa cells expressing sgNC or CYP4A11-targeting sgRNAs. Cells were pretreated with vehicle or 2 µM 17-HETE for 4 h, followed by treatment with vehicle, 150 nM RSL3 or 150 nM RSL3 plus 2 µM Lip-1 for an additional 24 h. z, Targeted eicosanoid metabolomics analysis measuring endogenous 17-HETE levels in HeLa cells expressing sgNC or CYP4A11-targeting sgRNAs after exposure to 200 nM RSL3 for 6 h. n = 3 biological replicates (c-f,h-l,o,q,s,v,w,y) and 6 independent samples (z). Data are mean ± s.d., analyzed by two-sided Student’s t-tests (z), one-way ANOVA (k,o) or two-way ANOVA (c,d,f,h-j,l,q,s,v,w,y). NS, not significant. Schematic in l created in BioRender; Dai, X. https://biorender.com/8vlxn8n (2026).
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