Fig. 3: (R)-CRZ and (S)-CRZ inhibit ferroptosis not through direct antioxidant activity or by affecting iron level.

A (S)-CRZ (1 μM) and (R)-CRZ (1 μM) inhibit RSL3 (0.25 μM)-induced lipid-ROS accumulation in 786-O cells. Cellular lipid-ROS was measured by C11-BODIPY 581/591 (1 µM) staining coupled with flow cytometry analysis. Fer-1 (1 μM) was used as a positive control for lipid-ROS elimination. B Both (S)-CRZ (50 μM) and (R)-CRZ (50 μM) have no antioxidant capacity in vitro by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay. The antioxidant capacity of indicated compounds was measured by scavenging DPPH. Trolox (50 μM) and Fer-1 (50 μM) were used as positive controls. C Both (S)-CRZ and (R)-CRZ have no antioxidant capacity in vitro by ABTS test. Each compound in this ABTS test was used at 1 mM. D Both (S)-CRZ and (R)-CRZ do not suppress the autoxidation of liposomes. Autoxidation of C11-BODIPY 581/591 (1 µM) and liposomes of egg phosphatidyl-choline lipids (1 mM) suspended in PBS (pH 7.4) was initiated by adding 1 mM AAPH. Indicated compounds (1 µM) were added to the reaction individually, and fluorescence signal of C11-BODIPY 581/591 was monitored. E (S)-CRZ and (R)-CRZ do not affect GSH and GSSG levels substantially. HT-1080 cells were treated with RSL3 for 2.5 h to induce ferroptosis. (S)-CRZ (3 μM) and (R)-CRZ (3 μM) were used in these experiments. GSH, GSSG and total (GSH + GSSG) level were measured as described in the Methods. F (S)-CRZ (3 μM) and (R)-CRZ (3 μM) treatment do not change cellular iron levels. Iron levels were measured by a Fe²⁺ indicator FerroOrange (1 μM). DFO (100 μM) was used as a positive control. G (S)-CRZ (3 μM) and (R)-CRZ (3 μM) do not affect the expression of genes involved in iron metabolism. Protein expression levels were validated by Western blot. Data in B–F are presented as the mean ± S.D., n = 3 independent repeats.