Fig 2: Morphine and iron upregulate FHC and FLC in cortical neurons. A, Iron-loading upregulates FHC and FLC in neurobasal cultures. Cultures were iron-loaded with FAC (25 µM) for 30 min, 6 h, or 24 h before lysis. Additionally, a negative control culture was iron-chelated with DFO (100 µM, 24 h) before lysis. Iron loading with FAC significantly increased FHC and FLC but only after 24 h. FHC and FLC expression were not significantly different at any time after treatment; N = 3 experiments; treatment F(4,20) = 12.94, p < 0.0001; FHC/FLC expression F(1,20) = 0.0029, p = 0.9576. B, Morphine upregulates FHC and FLC in neurobasal cultures. Cultures were treated with morphine (1 µM) or vehicle and lysed 30 min, 6 h, or 24 h after treatment. Morphine upregulated both FHC and FLC, but FHC was significantly upregulated at 6 h, while FLC reached significance at 24 h. However, the overall expression of FHC was not significantly different from FLC at each time point; N = 4 experiments; treatment F(3,24) = 22.94, p < 0.0001; FHC/FLC expression F(1,24) = 9.252, p = 0.0056. C, Morphine-treated rats upregulate FHC and FLC in frontal cortex tissue. Three-week-old Holtzman rats were treated with extended-release morphine or placebo pellets for 96 h as described in Figure 4 and the Materials and Methods. After the treatment, rats were killed and frontal cortex tissue was dissected, homogenized, and analyzed by Western blotting. Morphine significantly increased FHC and FLC expression in vivo, similarly to the in vitro experiment in panel B; N = 4 rats per treatment group; each column contains a homogenate from a different rat; treatment F(1,12) = 43.94, p < 0.0001; FHC/FLC expression F(1,12) = 3.814, p = 0.0745. All data were analyzed by two-way ANOVA and Tukey post hoc.

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: Ablation of Ncoa4 in cardiomyocytes showed defective ferritinophagy.(A) Western blot analysis of FTH1 in Ncoa4+/+ and Ncoa4–/– hearts 1 week after transverse aortic constriction (TAC). GAPDH was used as the loading control. The right-hand graphs show the densitometric analysis. The average value for sham-operated Ncoa4+/+ hearts was set to 1 (biologically independent samples: n = 5). (B) mRNA expression of Fth1 in the heart 1 week after TAC. Gapdh mRNA was used as the loading control. The average value for sham-operated Ncoa4+/+ hearts was set to 1 (biologically independent samples: n = 5). (C and D) Immunofluorescence analysis of LC3B (green) and FTH1 (red) in the heart 1 week after TAC. The number of LC3B- and FTH1-positive dots is shown in (D). (E and F) Immunofluorescence analysis of LAMP2a (green) and FTH1 (red) in the heart 1 week after TAC. The number of LAPM2a- and FTH1-positive dots is shown in (F). In (C) and (E), images of the square in the merged images are shown at higher magnification in the right three columns. Scale bar, 5 µm. Arrows indicate double-positive dots. The data were evaluated by one-way analysis of variance (ANOVA), followed by Tukey–Kramer’s post hoc test. *p<0.05, **p<0.001, ***p<0.0001. NS, p>0.05. Exact p-values are provided in Supplementary file 1.Figure 3—source data 1.Source data for Figure 3.

Fig 5: Ferroptosis of patient iHep is accompanied with degradation of ferritin in lysosome. A) Cellular free iron levels of control, patient and corrected iHep after treatment with or without 5 × 10−3 m FAC for 48 h. B) Western blotting of FTH1 in control, patient and corrected iHep after treatment with or without 5 × 10−3 m FAC for 48 h (left), with or without FAC for 48 h plus 50 × 10−6 m Baf A1 treatment (right). C,D) Representative confocal images of ferritin (green) and lysosomes (red) in control, patient, and corrected iHep after treatment with 5 × 10−3 m FAC for 48 h (left). Scale bar, 10 µm. The colocalization ratio of ferritin and lysosomes was shown in Pearson's correlation analyzed by image J in bar graph format from three independent experiments (right) (n = 3, 60 cells per group with three biological replicates). E) Relative lysosomal activity of control, patient and corrected iHep after treatment with or without 5 × 10−3 m FAC for 48 h (n = 3, 60 cells per group with three biological replicates). Representative images of this experiment are shown in Figure S10 (Supporting Information). F,G) Representative confocal images of ferritin (green) and NCOA4 (red) in control, patient, and corrected iHep after treatment with 5 × 10−3 m FAC for 48 h (left). Scale bar, 10 µm. The colocalization ratio of ferritin and NCOA4 was shown in Pearson's correlation analyzed by image J in bar graph format from three independent experiments (right) (n = 3, 60 cells per group with three biological replicates). These data are represented as mean ± SD (n = 3) and statistics was assessed using one‐way ANOVA D,G) and two‐way ANOVA A,E) followed by post‐hoc Holm–Sidak test, ***p < 0.001.