Evaluating ferroptosis susceptibility by monitoring lipid peroxidation in endoplasmic reticulum with a tailored...

Evaluating cellular susceptibility to ferroptosis, a regulated cell death process driven by the lipid peroxidation (LPO), is critical for understanding relevant pathogenesis and developing targeted therapies. This objective may be achieved by monitoring LPO within endoplasmic reticulum (ER), which is the earliest site of lipid peroxide accumulation during ferroptosis; however, tools for real-time and in situ detection of LPO in ER remain lacking. Herein, we report an ER-targeting fluorescence probe ER-LPO for monitoring LPO in ER and evaluating ferroptosis susceptibility. ER-LPO exhibited a sensitive, selective and rapid fluorescence off-on response at 518 nm to lipid peroxides, and could monitor both lipoxygenase-catalyzed enzymatic and hydroxyl radical-triggered non-enzymatic LPO. Using ER-LPO imaging, dynamic accumulation of lipid peroxides on the ER membrane was monitored during ferroptosis, which was negatively correlated with cell viability but independent of the induction pathways and cell lines. Notably, increasing polyunsaturated fatty acids (PUFA) in membrane phospholipids through PUFA supplementation or enhancing ACSL4-catalyzed PUFA assimilation accelerated LPO process and heightened cellular susceptibility to ferroptosis. Conversely, supplementing monounsaturated fatty acids or inhibiting ACSL4 to block the PUFA uptake could reduce LPO levels and facilitate ferroptosis resistance of cells. Moreover, ER-LPO enabled high-throughput screening of anti-ferroptosis reagents, identifying baicalein (a trihydroxyflavone derivative) as a potent ferroptosis inhibitor, which may be attributed to its intrinsic antioxidant capacity and lipoxygenase inhibitory activity. As an efficient tool for assessing ferroptosis susceptibility by monitoring of LPO in ER, ER-LPO may offer broad applications in mechanism studies, therapeutic evaluations and new drug developments for ferroptosis-related diseases.