Fig. 1.

Cysteine is capable to inhibit ferroptosis independent of GSH synthesis.

(A) Impact of cystine (CC) on glutathione (GSH) and oxidized glutathione (GSSG) level. MEFs were treated with indicated concentrations of CC for 8 h and subjected to assessment of GSH and GSSG level. (B and C) Analysis of lipid ROS level and cell death upon CC deprivation. MEFs were cultured in the absence or presence of CC (400 μM) for 8 h for evaluation of lipid ROS (B) or for 12 h for cell death detection (C). The lipid ROS accumulation or cell death were assessed by BODIPY 581/591C11 or propidium iodide (PI) staining coupled with flow cytometry analysis, respectively. Ferrastain-1 (Fer-1, 10 μM) and deferoxamine (DFO, 80 μM), two classical inhibitors of ferroptosis, were included as negative control. (D and E) Measurement of glutathione (GSH) and oxidized glutathione (GSSG) level upon impairment of GSH synthesis by l-buthionine sulfoximine (BSO). HT1080 cells (D) and MEFs (E) were cultured in the absence or presence of indicated amount of BSO for 24 h and subjected to assay of GSH and GSSG level. (F–I) Assay of lipid ROS level and cell death in the presence of BSO. Following treatment as described in Fig. 1 (D and E), HT1080 cells and MEFs were collected for assessment of lipid ROS (F and G) and cell death (H and I) as described in Fig. 1 (B and C). (J and K) Assessment of lipid ROS and cell death for GCLC-depleted cells. HT1080 cells were transfected with siRNA against GCLC or scramble for 60 h and subjected to measurement of lipid ROS (J) as described in Fig. 1 (B), and detection of cell death (K) as described in Fig. 1 (C). (L and M) Evaluation of intracellular cysteine level upon GSH inhibition. HT1080 cells and MEFs were cultured in the absence or presence of BSO (0.2 mM) for 24 h and subjected to detection of intracellular cysteine level. Data represent as mean ± SD of three independent experiments, with significance determined by one-way ANOVA test or t text. ***P＜0.001; ****P＜0.0001; ns, nonsignificant.