Fig. 1. Investigating the role of lipid-derived electrophile (LDE) detoxification in ferroptosis. (A) LDE and truncated phospholipid (PL) accumulation is a promising link between lipid hydroperoxide production and cell death in ferroptosis. Key relevant steps in ferroptosis involve high levels of lipid oxidation following induction through either GPX4 inhibition (RSL3) or GSH depletion (BSO). Iron chelation (DFO) or radical-trapping antioxidants (RTAs) prevent ferroptotic cell death. (B) LDE detoxification occurs through three steps: (1) GSH biosynthesis (inhibited by BSO), (2) GSH conjugation catalyzed by GST, and (3) active export through MRP channels (inhibited by sodium orthovanadate). (C) Widefield fluorescence imaging with AcroB enables monitoring of electrophile detoxification ability through quantification of both cellular fluorescence (fluorogenic AcroB retention, reporting on intracellular electrophile conjugation and level of intracellular electrophile adducts) and background fluorescence (AcroB active export/excretion, reporting on cellular electrophile adduct export). Widefield fluorescence image of a HeLa cell 15 minutes after addition of 100 nM AcroB, white line indicates cell border determined from complementary DIC image. Image acquired at 100× magnification, λ_ex = 488 nm (0.05 mW), scale bar is 12 μm, LUT range 0–5000.