Extended Data Fig. 2.

Tubastatin A did not affect the mRNA and protein level of GPX4.

(a–d) MDA-MB-231 or MCF-7 cells were treated with Tub or RSL3 at the indicated concentrations and times. The cells were collected and co-immunoprecipitation assays were used to isolate endogenous GPX4 from the cells. (e) Co-immunoprecipitation assays were used to isolate endogenous GPX4 from MDA-MB-231 or MCF-7 cells. (f–i) The relative GPX4 mRNA measurement in MDA-MB-231 cells (f, g) or MCF-7 cells (h, i) treated with Tub at the indicated concentrations and times. (j, k) The relative GPX4 protein measurement in MDA-MB-231 cells (j) or MCF-7 cells (k) treated with Tub at the indicated concentrations and times. f-i, statistical analysis was performed using an unpaired two-tailed Student's t-test. (l–o) Relative GPX4 enzyme activity measurement in the indicated test tube treated with CAY (CAY10603) or SKLB (SKLB-23bb) for the indicated concentration and time in cell free system. We used co-immunoprecipitation to isolate endogenous GPX4 from MDA-MB-231 cells. l, time: 2 h m, concentration: 8 μM. n, time: 2 h o, concentration: 8 μM. Data are the mean ± s.d.; n = 3 biologically independent experiments. Statistical analysis was performed using an unpaired two-tailed Student's t-test. a-e, j, k, Data are representative of n = 3 biologically independent experiments.