Extended Data Fig. 3: GPX4-R152 methylation promotes ferroptosis resistance in vitro.

a-c, Parental HeLa cells and the indicated clones with knock-in expression of GPX4-R152K mutant, were treated with or without cystine deprivation, Erastin (20 μM) and RSL3 (2 μM) for 6 h in the absence or presence of pretreated with Fer-1 (2 μM) for 2 h. Basal ROS levels (a), lipid ROS levels (b), and relative MDA levels (c) were measured. d-f, HeLa GPX4-knockout cells transfected with wild-type or the R152K mutant Flag-GPX4, were treated with or without cystine deprivation, Erastin (20 μM), and RSL3 (2 μM) for 6 h in the absence or presence of pretreated with Fer-1 (2 μM) for 2 h. Basal ROS levels (d), lipid ROS levels (e), and relative MDA levels (f) were measured. g-h, C4-2 GPX4-knockdown cells transfected with wild-type or the R152K mutant Flag-GPX4, were treated with or without cystine deprivation, Erastin (10 μM), and RSL3 (1 μM) for 6 h in the absence or presence of pretreated with Fer-1 (2 μM) for 2 h. Basal ROS levels (g) and lipid ROS levels (h) were measured. i-j, HeLa GPX4-knockout cells transfected with wild-type or the R152K mutant Flag-GPX4 (i), C4-2 GPX4-knockdown cells transfected with wild-type or the R152K mutant Flag-GPX4 (j), were treated with indicated concentrations of IKE and RSL3 for 24 h. Cell death were measured. k, Parental HeLa cells and the indicated clones with knock-in expression of GPX4-R152K mutant, were treated with or without Erastin (20 μM) and RSL3 (2 μM) for 24 h, then cultured for 2 weeks. Relative colony number were measured. l, HeLa GPX4-knockout cells transfected with wild-type or the R152K mutant Flag-GPX4, were treated with or without Erastin (20 μM) and RSL3 (2 μM) for 24 h, then cultured for 2 weeks. Relative colony number were measured. For a-l, data are mean ± s.d. of n = 3 biological replicates, P values were calculated using a two-tailed Student’s t-test. All of the experiments were repeated at least twice independently, yielding identical outcomes.

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