Figure 3.

Arginine deprivation protects cells against ferroptosis through reducing fumarate biosynthesis. (A) MEF cells were deprived of arginine for the indicated time (0, 2, 4, 6 h), the protein levels of GPX4, NRF2, and SLC7A11 were checked. (B) The expression of the NRF2 target genes was determined by RT-qPCR. (C) The diagram shows that fumarate is produced through the arginine-fueled urea cycle. ARG1: arginase 1, OTC: ornithine transcarbamylase. (D) The diagram shows that fumarate binds to GSH to generate succinicGSH. (E) MEF cells were incubated in the arginine-depleted medium for 6 h, and then the fumarate concentration was measured by HPLC. (F) Cells were co-treated with DMF (0, 1, 3, 10 µM) and 2.5 µM erastin with or without Fer in the arginine-depleted medium. Cell death was analyzed by PI staining followed by microscopy imaging and flow cytometry analysis. The representative images and the PI positive ratio are shown. Scale bar, 100 µm. (G) MEF cells were treated with the indicated concentration of DMF (0, 3, 5, 10 µM) in the arginine-depleted medium, and then the intracellular GSH was measured. (H,I) Cells were co-treated with DMF (10 µM) and 2.5 µM erastin with or without Fer in the arginine-depleted medium. Lipid ROS was determined with C11-BODIPY 581/591 staining followed by flow cytometry analysis and confocal microscopy imaging. The representative histogram of flow cytometry, relative mean fluorescence intensity, and representative images were shown. Scale bar, 10 µm. All data represent the mean ± SEM from three independent experiments. ** p < 0.01, ns, not significant. +Arg: complete medium, −Arg: arginine-depleted medium, Era: erastin, Fer: Ferrostatin-1.