Fig 2: The E3 ligase RNF8 degrades FAD-free FSP1.a, Schematic of sensitized UBAL degradation CRISPR–Cas9 screen. b, Gene effects and gene scores calculated for individual genes analyzed in the targeted degradation screen. c, STRING network map of the identified genes with a 10% FDR cutoff, where each bubble color represents an enriched GO term functional cluster. d, Fluorescence histograms of FSP1KO cells expressing FSP1–GFP in the indicated cell lines. e, Quantification of MFI change in GFP from d. Data are shown as the median ± s.e.m. of four independent experiments. f, Immunoblot of lysates from d. g,h, Ubiquitinated FSP1–GFP conjugates affinity-captured from lysates treated with 1 µM MG132 for 24 h (g) or expressing exogenous RNF8 variants (h). i, Kinetics of GFP fluorescence decay of FSP1KO cells expressing FSP1–GFP in Cas9 control or RFKKO cells with or without the loss of RNF8 following Dox washout. j, FSP1–GFP or RNF8 S-tag immunoprecipitation from FSP1KO cells expressing FSP1–GFP in the indicated cell lines and blotting for RNF8 (top) or FSP1 (middle). k, Model illustrating the mechanism by which vitamin B2 metabolism and FAD synthesis impact FSP1 activity and stability to prevent ferroptosis.Source data

Fig 3: RNF8 targets FAD-free FSP1 for degradation.a, Cloud plots indicating count numbers (top) and distribution for the relative enrichment (bottom) corresponding to RNF8 (color scale) and control (gray scale) sgRNAs. b-c, Fluorescence histograms of FSP1GFP-P2A-BFP cells in the indicated cell lines showing changes in GFP:BFP ratio (b) or GFP and BFP fluorescence (c). Data are shown as the median ± SEM of 3 independent experiments. d, Immunoblot of FSP1GFP-P2A-BFP lysates from (b-c). e, Alpha-fold superposition of RNF8 (O76064, red) and CHIP (Q9UNE7, gray) monomers. Close-up highlights how the RNF8 RING domain resembles that of the CHIP U-box domain. Source data