Classically, ferroptosis is triggered in vivo by inhibiting cellular antioxidant systems, mainly

GPX4 (Seiler et al, 2008). Inhibition of GPX4 leads to the accumulation of (phospho)lipid

hydroperoxides (PLOOH). However, to initiate lipid oxidation, free radicals are required. One

of the hypotheses is that free radicals initiating lipid oxidation are produced from hydrogen

peroxide (H2O2). However, it is not clear if H2O2 is produced upon ferroptosis induction e.g. by

the knockout of GPX4. Another important question in understanding ferroptosis is where in

the cell and which free radicals are triggering lipid oxidation.To answer these questions, we propose

to use a chemogenetic system, based on ascorbate peroxidase (APEX2).

APEX2 is an engineered heme peroxidase that can be stably expressed

in cells (Lam et al., 2015). APEX2 utilizes H2O2 to oxidize a substrate in one-electron steps.

Depending on the substrate provided, APEX2 can be used to either to detect H2O2 with

chromogenic, fluorogenic or luminogenic substrates or generate free radicals.