Fig 2: IRP2 regulation by ISC deficiency.Model for IRP2 activation in ISC deficiency and subsequent ferroptosis sensitivity. At atmospheric O2 levels, the FBXL5 ISC is oxidized, and IRP2 is ubiquitinated and degraded. Partial inhibition of ISC synthesis by shNFS1 or a dominant negative ISCU (mISCU) abrogates the FBXL5:IRP2 interaction, leading to increased IRP2 stability and IRP2 binding to target mRNAs. At physiologic O2 levels, the FBXL5 ISC is reduced, and degradation of IRP2 is limited. Partial inhibition of ISC synthesis does not further alter IRP2 stability but still releases IRP2 from FBXL5. ISC inhibition enhances IRP2 binding to target mRNAs independent of FBXL5 binding. IRP2 activation increases intracellular Fe+2 pool and sensitize cells to ferroptosis. Deletion of both IRP1 and IRP2 is sufficient to inhibit the iron accumulation and subsequent ferroptosis sensitivity.

Fig 3: IRP1/2 double-knockout cells expressing TFR1 can proliferate without iron supplementation and are sensitive to ferroptosis, but cannot sense ISC deficiency and enhance ferroptosis sensitivity.(A) Proliferation (relative cell number) of MDA-MB-231 cells expressing dox-repressible IRP2, TFR1 open reading frame (TFR1 without IRE) with 100 nM dox as indicated. (B) Immunoblots of IRP1/IRP2 double-knockout MDA-MB-231 cells expressing dox-repressible IRP2, TFR1 open reading frame, and nontargeting shRNA (shGFP, −) or shRNA targeting NFS1 (shNFS1, +), treated with 100 nM dox. (C) Relative viability of cells as in (B), treated with 100 nM dox, 1 μM ferrostatin, or erastin (0, 2.5, 5, and 10 μM) for 2 days as indicated. (D) Immunoblots of MDA-MB-231 cells expressing dox-repressible IRP2, TFR1 open reading frame, and shGFP or shRNAs targeting FXN (shFXN, 1 or 2), treated with 100 nM dox. (E) Relative viability of cells as in (D), treated with 100 nM dox, 50 μM Trolox, or erastin (0, 2.5, 5, and 10 μM) for 2 days as indicated. Asterisks indicate significance comparing shGFP to shNFS1 or shFXN for each group. P < 0.05, error bars are SEM.

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: IRP1 targets Bcl-xL to modulate mitophagy. (A and B) HeLa cells with stable tetracycline (Tet)-induced Bcl-xL expression were pre-treated with Tet or the vehicle (V) for 6 h, and then incubated with DFO for 24 h (A) or transfected with si-ISCU or NC siRNA for 72 h (B). The cells were harvested and analyzed by immunoblotting. The relative levels of indicated bands are shown in the right histograms. Data represent the mean ± SEM of three independent experiments. **p < 0.01. (C and D) HeLa cells stably co-expressing mt-Keima and Tet-induced Bcl-xL were pre-treated with Tet or the vehicle (V) for 6 h, then incubated with DFO for 24 h (C) or transfected with si-ISCU or NC siRNA for 72 h (D). The cells were collected and signals from green and red channels were detected by flow cytometry. The percentage of mitophagic cells with higher red/green fluorescence was showed in the below histograms as mean ± SEM from three experiments; **p < 0.01. (E) HeLa cells stably expressed sc shRNA or sh-IRP1 were transiently transfected with si-Bcl-xL or NC siRNA. After 48 h of transfection, the cells were challenged with DFO for 24 h. The cells were harvested and analyzed by immunoblotting. The relative levels of TIM23 are shown in the right histograms. Data represent the mean ± SEM of three independent experiments. **p < 0.01. (F) HeLa cells stably expressed sc shRNA or sh-IRP1 were transiently transfected with si-Bcl-xL accompanied with si-ISCU or the NC siRNA. After 72 h of transfection, the cells were harvested for immunoblotting analysis. The relative levels of TOM20 are shown in the right histograms. Data represent the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01.