Fig. 3.

Tubastatin A induces ferroptosis by directly inhibiting the enzymatic activity of GPX4. (a) (left) Schematic of biotin-streptavidin pulldown method: MDA-MB-231 cells were treated with biotin or biotin-linked Tub, and Tub-binding proteins were enriched on streptavidin beads, subjected to an on-bead trypsin digestion and subsequent LC/LC–MS/MS analysis. (right) Enrichment of proteins based on coverage and area. GPX4 was a top target candidate. (b–i) Relative GPX4 enzyme activity measurement in the indicated test tube treated with Tub or RSL3 for the indicated concentration and time in cell free system. b, c, f, g, we used co-immunoprecipitation to isolate endogenous GPX4 from MDA-MB-231 cells. d, e, h, i, we used co-immunoprecipitation to isolate endogenous GPX4 from MCF-7 cells. b, time: 2 h c, concentration: 8 μM. d, time: 2 h e, concentration: 8 μM. f, time: 2 h g, concentration: 8 μM. h, time: 2 h i, concentration: 8 μM. (j–q) Relative GPX4 enzyme activity measurement in the indicated cell treated with Tub or RSL3 for the indicated concentration and time. j, time: 20 h k, concentration: 8 μM. l, time: 20 h m, concentration: 8 μM. n, time: 20 h o, concentration: 6 μM. p, time: 20 h q, concentration: 6 μM. (r–w) Cell viability measurements in the indicated cells subjected to the indicated treatments for 28 h (r–t) or 36 h (u–w). b-q, Data are the mean ± s.d.; n = 3 biologically independent experiments. Statistical analysis was performed using an unpaired two-tailed Student's t-test. r-w, Error bars are mean ± s.d., n = 3 independent repeats. Statistical analysis was performed using a two-way ANOVA analysis.