Fig 2: Erastin treatment differentially affects TFRC and ferritin heavy chain 1 (FTH1) protein expression in cells expressing distinct TP53 mutation types. Representative images and quantification of Western blots examining the expression of (A,B) transferrin receptor 1 (TFRC) and (C,D) ferritin heavy chain 1 (FTH1) in TP53 null H1299 cells (H1299) or H1299 cells transfected with wild-type (WT) TP53, or the indicated TP53 mutant following treatment with DMSO (control) or 10 µM erastin for 24 h (n = 3–4 per cell type and treatment). Relative expression was normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. * Denotes statistical differences from respective control, p < 0.05. Error bars indicate SEM.

Fig 3: IRP2 is sufficient to sense ISC deficiency and sensitize cells to ferroptosis.(A) Proliferation (relative cell number) of IRP1/2 double-knockout MDA-MB-231 cells grown in media supplemented with 35 μM iron nitrate and 50 μM Trolox or upon withdrawal of supplements at day 0. (B) Immunoblots for indicated proteins of lysates derived from IRP1 knockout and IRP1/2 double-knockout MDA-MB-231 cell line treated with 50 μM DFO and 35 μM iron nitrate (1 day) as indicated. (B and C) Immunoblots for indicated proteins of lysates derived from parental (WT) and IRP1/2 double-knockout MDA-MB-231 cell line expressing a nontargeting shRNA (shGFP, −) or shRNA targeting NFS1 (shNFS1, +). (D) Relative abundance of TFR1 and FTH1 mRNA in parental, IRP1 knockout, and IRP1/2 double-knockout MDA-MB-231 cell line expressing shGFP or shNFS1. (E and F) Immunoblots for indicated proteins of lysates derived from IRP1/IRP2 double-knockout MDA-MB-231 cells expressing dox-repressible IRP2 treated with 100 nM dox for 2 days, and then treated with 100 nM dox, 50 μM DFO, or 35 μM iron nitrate (1 day), or infected with shGFP or shNFS1. After drug selection for shRNA transduction, cells are treated with 100 nM dox and 35uM iron nitrate for 1 day. (G) Relative viability of cells as in (E) and (F). After drug selection for shRNA transduction, cells were treated with 100 nM dox, 1 μM ferrostatin, or erastin (0, 2.5, 5, and 10 μM) for 2 days as indicated.

Fig 4: IRP1 knockout cells activate the iron-starvation response upon inhibition of ISC synthesis and become sensitive to ferroptosis.(A) Relative viability of parental (WT) and IRP1 knockout MDA-MB-231 cell line expressing a nontargeting shRNA (shGFP, −) or NFS1 (shNFS1, +), treated with erastin (0, 2.5, 5, and 10 μM; 2 days). (B) Labile iron pool of parental and IRP1 knockout MDA-MB-231 cell line expressing shGFP or shNFS1. (C) Immunoblots for indicated proteins from lysates derived from parental (WT) and IRP1 knockout MDA-MB-231 cell line expressing shGFP or shNFS1, treated with 50 μM Deferoxamine (DFO) and 35 μM iron nitrate as indicated (1 day). (D) Relative abundance of TFR1 and FTH1 mRNA in parental and IRP1 knockout MDA-MB-231 cell line expressing shGFP or shNFS1. (E) Relative viability of WT and IRP1 knockout 293 human embryonic kidney (HEK) cell line expressing dox-inducible ISCU-DN treated with 100 nM dox (1 day) and then treated with 100 nM dox, 1 μM ferrostatin (Fer.), and erastin (Eras.; 0, 2.5, 5, and 10 μM; 2 days) as indicated. (F) Immunoblots for indicated proteins of lysates derived from parental and IRP1 knockout HEK-293 cell line expressing dox-inducible ISCU-DN treated with dox (0, 10, and 100 μM; 1 day) as indicated. (G) Immunoblots for indicated proteins of lysates derived from parental and IRP1 knockout MDA-MB-231 cells expressing GFP reporter with 5′UTR IRE sequence from FTH1 mRNA and shRFP or shNFS1. *P < 0.01; error bars are SEM.

Fig 5: Ferritinophagy induced by NDV contributes to ferroptosis initiation(A) Western blot analysis of the levels of LC3 and NCOA4 in NDV-infected U251 cells. Baf-A1 was used as an autophagy inhibitor. β-actin was used as the loading control.(B) Fluorescence microscopy was used to colocalize NCOA4 (red) and LC3 (green) in U251 cells. Rapamycin was used as an autophagy inducer. Scale bars = 20 μm.(C) Western blotting analysis of the expression levels of TFR1 in NDV-infected U251cells. Western blotting samples corresponding to the marked timepoints were collected and analyzed.(D and E) FTH1 and NCOA4 expression levels in NCOA4-knockdown cells. Cells were treated for 24 h with or without NDV. Data are representative of three experiments.(F) Expression levels of Reactive oxygen species were observed in U251 cells with NCOA4 knockdown with fluorescence microscopy, Erastin was used as a ferroptosis inducer.(J and H) Expression levels of ferrous iron in NCOA4-knockdown cells were observed with confocal fluorescence microscopy, Erastin was used as a ferroptosis inducer.(I) Cell death was examined with an LDH assay 24 h after pretreatment with or without DFO.(J) A model of the possible mechanisms underlying the induction of ferroptosis by NDV through nutrient deprivation and ferritinophagy in U251 cells.Significance was analyzed using a two-tailed Student's t-test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Data were expressed as mean ± SEM (n = 3 in each group).