Fig. 1: USP24 is downregulated during ferroptosis induced by GPX4 inhibitors.

A, B MDA-MB-231 cells were exposed to 0.5 μM RSL3 for varying time intervals or to different concentrations of RSL3 for 12 h. Subsequently, cellular protein expression was assessed by western blot analysis. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. C, D MDA-MB-468 cells were treated with the indicated doses of RSL3 for 12 h, and then cellular proteins were analyzed with western blotting. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD, n = 3. ***P < 0.001, ****P < 0.0001, ns no significance. E, F T47D and MCF-7 cells were treated with the indicated doses of RSL3 for 12 h, and then cellular proteins were analyzed with western blotting. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. ns no significance. G, H MDA-MB-231 cells were treated with the indicated concentration gradient of ferroptosis inducers, including RSL3, ML162, ML210, FIN56, and erastin for 12 h, and then USP24 proteins were analyzed with western blotting. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. *P < 0.05, **P < 0.01. ns no significance. I, J MDA-MB-231 cells were treated with the indicated concentration gradient of ferroptosis inducers, including RSL3, ML162, ML210, FIN56, and erastin for 12 h, and then ubiquitinated proteins (Ub) were analyzed with western blotting. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0. 001, ****P < 0.0001. ns no significance. K, L Western blotting was used to examine the effects of ferroptosis inhibitor ferrostatin-1 (Fer-1, 2 μM) on RSL3 (0.5 μM for MDA-MB-231 cells; 0.25 μM for MDA-MB-468 cells, 12 h)-induced ubiquitination (Ub) accumulation and USP24 downregulation. Quantification of protein levels, normalized to β-actin, was performed based on three independent experiments. Data were presented as Mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001.