Extended Data Fig. 5: Characterization of Lysine 48 mutants of GPX4 in cells and in vitro.

a, Total GPX4 activity of HT1080 cells overexpressing GFP-GPX4^WT, GFP-GPX4^K48A, or GFP-GPX4^K48L and a control line. Data are plotted as means ± SD of eleven biologically independent replicate experiments. Ordinary one-way ANOVA followed by Tukey’s multiple comparisons test was performed and P values were plotted, n = 11, DF = 40. b, HT1080 overexpressing exogenous WT, K48A, or K48L GFP-GPX4 and a control line were tested for RSL3 sensitivity. Data are plotted as means ± SD of three biologically independent replicate experiments. c-d, SDS-PAGE gel of His-tagged GPX4^U46C_K48A and GPX4^U46C_K48L as stained by Coomasie Blue. Biologically independent duplicate (GPX4^U46C_K48A) and quadruplicate (GPX4^U46C_K48L) experiments were performed and imaged. e, The distances between catalytic residues Sec46 and Trp136 were recorded every 4.8 ps throughout MD simulations of GPX4^WT (PDB: 6HN3), GPX4^K48A, GPX4^K48L, GPX4^K48E, GPX4^K48Q, and GPX4^K48R. Representative data from three times 100 ns trajectories were plotted as means ± SD. Ordinary one-way ANOVA followed by Tukey’s multiple comparisons test was performed: n = 20835, DF = 125004, all P**** < 1x10^-20. f, Scheme illustrating the catalytic cycle of sulfur-containing variant of GPX4.

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