Extended Data Fig. 3: Preparation of cell models of GPX4^R152H.

a, HT-1080 transfected with pBP GFP-cGPX4^WT, pBP GFP-cGPX4^R152H, pBabepuro (pBP) empty vector, pBP GFP-cGPX4^K48A, pBP GFP-cGPX4^K48L and pBP GFP-cGPX4^K125R-K127R were selected with puromycin and imaged with microscope. Triplicate experiments were repeated independently with similar results while the representative images were shown. The plotted scale bar is 400 µm. b, Total GPX4 enzymatic activity (endogenous apo-GPX4 and transfected exogenous GFP-tagged-GPX4) of control HT1080 (pBP, no expression of GFP-tagged-GPX4) and HT1080 cells overexpressing GFP-GPX4^WT or GFP-GPX4^R152H. Data are plotted as means ± SD of six replicate experiments. Ordinary one-way ANOVA followed by Tukey’s multiple comparisons test was performed: n = 6, DF = 15 and P values were plotted. c, Western blot of control HT1080 (pBP) and HT1080 cells overexpressing GFP-GPX4^WT or GFP-GPX4^R152H using GPX4 and GAPDH antibodies. Expression levels of GFP-WT-GPX4 and GFP-R152H-GPX4 were quantified. Data are plotted as means ± SD, n = 4 biologically independent samples. Unpaired two-tailed t test was then performed and plotted: t = 0.3158, df=6, P^ns = 0.7629. d, Western blot of Gpx4-knockout Pfa1 cells overexpressing exogenous human WT or R152H GPX4, Gpx4-knockout Pfa1 cells overexpressing exogenous murine WT or R152H mScarlet-tagged GPX4, and Gpx4-knockout HT1080 cells overexpressing exogenous murine WT or R152H mScarlet-tagged GPX4 using GPX4 and GAPDH antibodies. Expression levels of GPX4 were quantified. Data are plotted as means ± SD, n = 3 biologically independent samples. Ordinary two-way ANOVA followed by Sidak’s multiple comparisons test was performed and P values were plotted: n = 3, DF = 12. Full scan image is shown in the Supplementary Information.

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