Fig 2: PEITC altered iron metabolism in human OS cells. (a) Total iron level in MNNG/HOS, U-2 OS, MG-63, and 143B cells after the indicated concentrations of PEITC treatment for 24 h by AAS. (b) The level of labile iron in MNNG/HOS, U-2 OS, MG-63, and 143B cells after the indicated concentrations of PEITC treatment for 24 h with Calcein-AM staining by flow cytometry analysis. (c) Protein expression levels of TfR1, DMT1, FTH1, FPN, and IRP2 in MNNG/HOS, U-2 OS, MG-63, and 143B cells treated with the indicated concentrations of PEITC for 20 h or 30 μM PEITC for 4 h, 12 h, 24 h, and 48 h. (d) mRNA levels of TFRC, SLC11A2, FTH1, SLC40A1, and IRP2 in MNNG/HOS, U-2 OS, MG-63, and 143B cells after PEITC treatment for 24 h. (e) Protein expression levels of NCOA4 in MNNG/HOS, U-2 OS, MG-63, and 143B cells treated with the indicated concentrations of PEITC for 20 h or 30 μM PEITC for 4 h, 12 h, 24 h, and 48 h. All data were presented as the means ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus control group.

Fig 3: Disrupting V-ATPase activity decreases intracellular iron levels.(A, B) V-ATPase inhibition leads to intracellular iron depletion. (A) HeLa cells were treated with BafA (10 nM or 100 nM) or 100 µM DFO for 24 hr. HIF1α, IRP2, NCOA4 and ferritin (ferritin heavy chain 1, FTH1) levels were measured by immunoblot. (B) HIF1α-GFPODD reporter cells transduced with Cas9 and sgRNA targeting V-ATPase components (TMEM199, CCDC115, ATP6V0D1 and ATP6V1A1) were sorted into GFPLOW (Lo) and GFPHIGH (Hi) populations as described. The lysates were immunoblotted for HIF1α, IRP2, or NCOA4. β-actin served as a loading control. (C) Iron chelation prevents HIF1α hydroxylation. In vitro prolyl hydroxylation of the HIF1αODD protein following incubation with lysates from WT or DFO treated lysates (100 µM for 24 hr) as previously described. DMOG served as a control for PHD inhibition. (D–F) In vitro hydroxylation of PHD activity in DFO or BafA treated lysates supplemented with ferrous iron. Lysates from control, DFO (D) or BafA (E) treated HeLa cells were extracted as previously described, incubated with the HIF1αODD protein, and supplemented with increasing concentrations of iron chloride (FeCl2, Fe(II)). Prolyl-hydroxylated HIF1αODD levels were visualised by immunoblot and quantified by densitometry for the BafA treated lysate (F) (n = 3). Values are mean±SEM. *p<0.05, **p<0.01 Fe(II) compared to no treatment in BafA treated cells. NS=not significant.DOI: http://dx.doi.org/10.7554/eLife.22693.011

Fig 4: Suppression of ISC synthesis increases IRP2 stability independent of IRP1 in atmospheric O2 levels.(A and B) Immunoblots for indicated proteins of lysates derived from parental (WT) (A) and IRP1 knockout (B) MDA-MB-231 cells expressing a nontargeting shRNA (shGFP, −) or shRNA targeting NFS1 (shNFS1, +) incubated in 3% or 21% O2. (C and D) Immunoblots for indicated proteins from lysates derived from IRP1/IRP2 double-knockout MDA-MB-231 expressing dox-repressible IRP2 and shGFP or shNFS1, treated with 500 nM dox for 0, 1, 2, or 3 days in 21% (C) or 3% O2 (D).

Fig 5: 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.