Fig. 2. Cys106 of DJ-1 is involved in regulation of ferroptosis.

a Extracted ion chromatograms (EICs) of the IAyne labeled peptides containing C46 or C106 from DJ-1. The peptide EICs from the normal, ferroptotic and rescued cells are colored in red, yellow and blue, respectively. The quantified ratios are shown above. b Quantified ratios of DJ-1 comparing the protein abundance between the normal and ferroptotic cells or between the normal and rescued cells. c Immunoblotting shows selective labelling of DJ-1 in normal, ferroptotic and rescued cells by IAyne. IAyne failed to pull down DJ-1 from the ferroptotic cells. d, e Knockout of DJ-1 sensitized cells to ferroptosis that can only be reversed by the overexpression of the WT but not mutant DJ-1. Dose-dependent cell death induced by RSL3 were measured by MTT. DJ-1 knockout subclones (DJ-1 KO) were generated from HT1080 cells by CRISPR/Cas9 using corresponding guide RNAs. f, g Stable expression of the WT but not mutant DJ-1 exerts protection of cells from ferroptosis. Dose-dependent cell death induced by RSL3 were measured by MTT. Cells were stably transfected with DJ-1 WT/ C106A expression plasmids or empty vehicle (EV). h Knockout of DJ-1 increased the level of lipid ROS in ferroptotic cells that can only be suppressed by the overexpression of the WT but not the C106A mutant DJ-1. i Stable expression of WT but not the C106A mutant DJ-1 suppresses the level of lipid ROS in the early stage of ferroptosis. In h, i time-course measurement of lipid ROS in RSL3-induced ferroptotic cells were performed with the fluorescent probe C11-BODIPY by flow cytometry using FITC 510 nM. Error bars in b, d–i indicate standard deviations from triplicates. Data was analyzed by student’s t-test (^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001; n.s., not significant, n = 3).