Journal: bioRxiv

Article Title: Vitamin B2 metabolism promotes FSP1 stability to prevent ferroptosis

doi: 10.1101/2025.08.05.668752

Figure Lengend Snippet: 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 FSP1 KO cells expressing FSP1-GFP in the indicated cell lines. e , Quantification of median fluorescence intensity (MFI) change in GFP from ( d ). f , Immunoblot of lysates from ( d ). g-h , Kinetics of GFP fluorescence decay of FSP1 KO cells expressing FSP1-GFP in control or RFK KO cells with or without the loss of RNF8 ( g ) or RFK/RNF8 DKO (double knock-out) cells expressing the indicated RNF8 variants ( h ) following doxycycline washout. i , FSP1-GFP or RNF8 S-tag immunoprecipitation from FSP1 KO cells expressing FSP1-GFP in the indicated cell lines and blotting for RNF8 (top) or FSP1 (middle). j , Model illustrating the mechanism by which vitamin B2 metabolism and FAD synthesis impacts FSP1 activity and stability to prevent ferroptosis.

Article Snippet: For immunoprecipitation of FSP1 and RNF8 interactions, 1 mg of pre-cleared lysates were incubated with 10 μL of ChromoTek GFP-Trap Magnetic Agarose or 10 μL of S-protein Agarose (Millipore Sigma, no. 69704) for 1 h at 4°C with end-over-end rotation.

Techniques: CRISPR, Functional Assay, Fluorescence, Expressing, Western Blot, Control, Knock-Out, Immunoprecipitation, Activity Assay