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. 2016 Aug 12;26(9):1021–1032. doi: 10.1038/cr.2016.95

Figure 6.

Figure 6

NCOA4-mediated ferritinophagy promotes ferroptosis. (A, B) Ferroptosis induced NCOA4 degradation in a time-dependent manner in MEFs (A) and HT1080 cells (B). MEFs or HT1080 cells were treated as indicated. Total cell extract was used for western blot to detect NCOA4 change during ferroptosis. erastin: 0.5 μM (A) or 5 μM (B). (C, D) Autophagy inhibitor BafA1 can block ferroptosis-induced NCOA4 degradation in MEFs (C) and HT1080 cells (D). MEFs or HT1080 cells were treated as indicated. Total cell extract was used for western blot to detect NCOA4 change during ferroptosis. erastin: 0.5 μM (C) or 5 μM (D). BafA1: 20 nM. (E, F) Genetic disruption of autophagy blocks ferroptosis-induced NCOA4 degradation. ATG13KO and ATG13-reconstituted MEFs (E), or ATG3KO and ATG3-reconstituted MEFs (F), were treated with 1 μM erastin for the indicated times. Total cell lysate was used for western blot to detect NCOA4 change during ferroptosis. (G) Knockdown of NCOA4 by shRNAs can block ferroptosis. MEFs infected with non-target (NT) shRNA or two independent NCOA4 shRNAs were treated with 0.5 μM erastin for 12 h. Cell death was measured by PI staining coupled with flow cytometry. (H) Knockdown of NCOA4 by shRNA in MEFs can block ferroptosis-associated lipid ROS accumulation. NCOA4-knockdown or control knockdown cells were treated with 1 μM erastin for 10 h. Lipid ROS was measured by C11-BODIPY staining coupled with flow cytometry.