Extended Data Fig. 1: OGT is oxidized and activated by ROS produced during ferroptosis, related to Fig. 1.

a-d, PLC/PRF/5 cells were treated with erastin for the indicated time (a, b) and concentrations (c, d). Immunoblot analyses were conducted with indicated antibodies(a, c) and quantified by ImageJ (b, d) (n = 3 experiments). e. f, Huh7 cells were treated with the indicated concentrations of RSL3. Immunoblot analyses were conducted with indicated antibodies and quantified by ImageJ (n = 3 experiments). g, MS analysis was performed to identify the oxidation sites on OGT. HA-OGT was purified from HEK293T cells following H₂O₂ treatment and then analysed by mass spectrometry. h, Alignments of the oxidation motif in OGT across human and other species. i-k, Huh7 cells were treated with erastin (10 μM) for 24 h (i), RSL3 (500 nM) for 24 h (j), and H₂O₂ (100 μM) for 1 h (k). The intracellular ROS level was detected by flow cytometry analysis. n = 3 independent experiments. l. m, Huh7 cells were treated with the indicated concentrations of H₂O₂. Immunoblot analyses were conducted with indicated antibodies (l) and quantified by ImageJ (m) (n = 3 experiments). n, Endogenous OGT in Huh7 cells was depleted using shRNA, then rescued with either OGT-WT or OGT-C845S. Immunoblot analyses were conducted with indicated antibodies. b,d,f,i,j,k,m Data are presented as the mean ± SEM; two-tailed Student’s t-test.

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