Extended Data Fig. 8: GPX4 T40/S44 phosphorylation prerequisites GPX4 for binding with FBW7.

a, HEK-293T cells were transfected with indicated Flag-GPX4 constructs, Cell lysates and the immunocomplexes from anti-Flag immunoprecipitant were analyzed by western blotting using indicated antibodies. b, HEK-293T cells were transfected with HA-FBW7 together indicated Flag-GPX4 constructs, Cell lysates and the immunocomplexes from anti-HA immunoprecipitant were analyzed by western blotting using indicated antibodies. c, HEK-293T cells were transfected with HA-FBW7 together with indicated Flag-GPX4 constructs or His-Ub, Cell lysates and the immunocomplexes from Ni-NTA pulldown were analyzed by western blotting using indicated antibodies. d-g, HEK-293T cells were transfected with indicated Flag-GPX4, HA-FBW7, and Myc-Cullin1 constructs. Cell lysates were analyzed by western blotting using indicated antibodies. h, HEK-293T cells were transfected with HA-FBW7 together with Flag-GPX4 constructs followed by indicated kinase inhibitor treatment, Cell lysates and the immunocomplexes from anti-HA immunoprecipitant were analyzed by western blotting using indicated antibodies. i-m, shGPX4 C4-2 cells stably expressing wild-type, T40E/S44E (EE) or T40A/S44A (AA) GPX4 were subjected to mouse xenograft assays. When the tumor reached 50 mm³, the mice were assigned randomly into different treatment groups treat with or without IKE (100 mg/kg). Tumor sizes were monitored (i), and dissected tumors were weighed (j-k), Reprehensive image (l) and statistical analysis (m) of IHC staining with indicated antibodies of xenograft tumors from j were shown. Scale bar, 100 μm. Data in i, k and m represent the mean ± s.e.m., n = 5 mice. For i, P values were calculated using a two-tailed log-rank test. For k and m, P values were calculated using a two-tailed Student’s t-test. All of the experiments, from a to h, were repeated at least twice independently, yielding identical outcomes.

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