Article Figures & Data
Figures
- FIG 1
Iron deficiency disrupts mitochondrial respiratory chain complex assembly and reduces mitochondrial respiratory activity and membrane potential. (A) HeLa cells were incubated with 100 μM DFO for the indicated times and immunoblotted using anti-FTH1 and anti-FTL antibodies. (B) The postnuclear supernatant (PNS) fraction obtained from DFO-treated HeLa cells was subjected to BN-PAGE followed by immunoblotting. (C) OCRs in DFO-treated HeLa cells were measured by using an extracellular flux analyzer. Next, 1 μM oligomycin A, 1 μM FCCP, and 5 μM antimycin A with 10 μM rotenone were injected at the indicated times to determine the proportions of oxygen consumption due to ATP production and the basal and maximum rates of respiration. The results are from three independent experiments. (D) Basal respiration, ATP production, and maximum respiration were quantified. (E and G) Isolated mitochondria were prepared from HeLa cells treated with 100 μM DFO for 12 and 24 h. BN-PAGE and SDS-PAGE were performed using the indicated antibodies. Data are representative of results from three independent experiments. (F) Results of quantification of data in panel E from three independent experiments. (G) HeLa cells were treated with 100 μM DFO or 1 μM valinomycin for the indicated times and with anti-Tom20 and MitoTracker CMXRos. Bar = 10 μm. (H and I) Results of quantification of the fluorescence intensity ratios of MitoTracker Red CMXRos and Tom20 from 30 cells. Error bars represent mean values ± standard errors of the means (SEM). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Dunnett’s multiple-comparison test was performed for panel I).
- FIG 2
A lysosomal inhibitor reduces respiratory chain complex assembly and induces mitochondrial dysfunction. (A) HeLa cells were incubated with 100 μM DFO, 10 nM BAF, and 50 μg/ml E64d with pepstatin A for 24 h. Cell lysates were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. Data are representative of results from three independent experiments. (B) The PNS obtained from HeLa cells treated with 100 μM DFO and 10 nM BAF or 1 μM epoxomicin was subjected to BN-PAGE prior to Western blotting using the indicated antibodies. Data are representative of results from three independent experiments. (C) Cell lysates from HeLa cells incubated with 100 μM DFO, 100 μM FAC, 10 nM BAF, or 100 μM FAC for 24 h were immunoblotted with the indicated antibodies. Data are representative of results from three independent experiments. (D) OCRs in cells treated with 100 μM DFO, 10 nM BAF, or 10 nM BAF with 100 μM FAC for the indicated times were measured. Results are from five independent experiments. (E) Basal respiration, ATP production, and maximum respiration were quantified. (F and G) Mitochondria were isolated from HeLa cells treated under the indicated conditions. SDS-PAGE and BN-PAGE followed by immunoblotting were performed using the indicated antibodies. Error bars represent mean values ± SEM. **, P < 0.01; ***, P < 0.001 (a Tukey-Kramer test was performed for panel E).
- FIG 3
The importance of NCOA4 in maintaining mitochondrial function via ferritin complex degradation in lysosomes. (A) HeLa cells were transfected with siNCOA4 or scrambled siRNA. Cells were incubated with 100 μM DFO for 24 h and immunoblotted with the indicated antibodies. (B) The PNS of NCOA4 KD and control cells was subjected to BN-PAGE followed by immunoblotting with the indicated antibodies. (C) NCOA4 KD and control cells incubated with 100 μM DFO were stained with DAPI (4′,6-diamidino-2-phenylindole), anti-LAMP2, and anti-FTH1. Bar = 10 μm. (D) LAMP2-positive lysosomes that colocalized with FTH1 from 30 cells. (E and F) HeLa cells transfected with siNCOA4 or control siRNA (siControl) were incubated with 100 μM DFO for 12 h or 100 μM FAC for 24 h. Mitochondria isolated from these cells were subjected to SDS-PAGE and BN-PAGE followed by immunoblotting using the indicated antibodies. (G) OCRs in NCOA4 KD and control cells treated under the indicated conditions were measured. (H) Results of quantification of basal respiration, ATP production, and maximum respiration. The results are from five independent wells. (I) HeLa cells transfected with control siRNA and siNCOA4 were expressed with siRNA-resistant GFP-NCOA4 or GFP. Western blotting was performed using the indicated antibodies. (J) HeLa cells transfected with control siRNA and siNCOA4 were expressed with siRNA-resistant GFP-NCOA4 or GFP. Isolated mitochondria were prepared from these HeLa cells. BN-PAGE and SDS-PAGE were performed using the indicated antibodies. (K) OCRs in NCOA4 KD and control cells transfected with or without siRNA-resistant GFP-NCOA4 or GFP were measured. (L) Quantification of data in panel K, from three independent experiments. (M) HeLa cells transfected with siNCOA4 or siControl were expressed with pMTS-mCherry, in which the mitochondrial targeting sequence of human PINK1 was ligated into mCherr-N (30). These cells, incubated with or without 100 μM DFO for 24 h, were stained with 5 μM Mito-FerroGreen. (N) Results of quantification of the fluorescence intensity ratios of mCherry and Mito-FerroGreen from 30 cells. Error bars represent mean values ± SEM. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Tukey-Kramer tests and Dunnett’s multiple-comparison tests were performed for panels H and L and panels D and N, respectively).
- FIG 4
Autophagy contributes to ferritin complex degradation under iron-deprived conditions. (A) HeLa cells transfected with siFIP200 or siControl oligonucleotides were incubated with 100 μM DFO for the indicated times. OCRs were obtained from five independent wells. (B) Cells transfected with siFIP200 or siControl were treated with 100 μM DFO for 24 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. (C and D) Mitochondria and the PNS were obtained from FIP200 KD and control cells treated with 100 μM DFO for 24 h (C) and 12 h (D). BN-PAGE and SDS-PAGE followed by immunoblotting were performed with the indicated antibodies. (E) HeLa cells transfected with siFIP200 or siControl oligonucleotides were incubated with 100 μM DFO or 100 μM FAC for the indicated times. OCRs were obtained from five independent wells. Error bars represent mean values ± SEM.
- FIG 5
The ferritin light chain is required for iron supply to mitochondria under iron-deprived conditions. (A) Wild-type (WT) and FTL knockout (KO) HeLa cells treated with 100 μM DFO for the indicated times were immunoblotted with the indicated antibodies. (B) The PNS was obtained from WT and FTL KO HeLa cells treated with 100 μM DFO for 24 h. Shown are results from BN-PAGE followed by immunoblotting with the indicated antibodies. (C) Representative images of WT and FTL KO cells incubated with 100 μM DFO for 24 h and immunostained with anti-LAMP2 and anti-FTH1 antibodies. Bar = 10 μm. (D) WT and FTL KO HeLa cells expressing GFP-NCOA4 incubated with 100 μM DFO for 24 h. Immunostaining with anti-FTH1 antibodies was performed. Arrows indicate GFP-NCOA4 puncta that colocalized with FTH1. Bars = 10 μm. (E) FTL WT and KO cells expressing GFP-NCOA4 or GFP were incubated for 48 h. Cell lysates were subjected to immunoprecipitation (IP) using anti-GFP magnetic beads. Immunoprecipitates were analyzed by Western blotting. (F) OCRs in WT and FTL KO cells were measured. OCRs were obtained from four independent wells. (G) Results of quantification of basal respiration, ATP production, and maximum respiration. The results are from five independent wells. (H) Mitochondria were isolated from FTL WT and KO cells expressing FLAG-FTL or an empty vector incubated with 100 μM DFO for 6 h. BN-PAGE and SDS-PAGE followed by immunoblotting were performed with the indicated antibodies. (I) FTL KO cells, FLAG-FTL cells, or empty vector cells were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. (J and K) FTL KO cells were transfected with FLAG-FTL or an empty vector with (J) or without (K) GFP-NCOA4. Cells treated with 100 μM DFO for 24 h were immunostained with anti-LAMP2 and anti-FTH1 (K) or anti-FTH1 (J) antibodies. Bars = 10 μm. (L) OCRs in FTL KO cells transfected with FLAG-FTL or an empty vector were measured. OCRs were obtained from five independent wells. (M) Results of quantification of basal respiration, ATP production, and maximum respiration from panel L. Error bars represent mean values ± SEM. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Tukey-Kramer tests were performed for panels G and M).
Additional Files
Supplemental material
- Supplemental file 1 -
Fig. S1 (Lysosomal inhibitor reduces respiratory chain complex assembly and induces mitochondrial dysfunction)
PDF, 5.8M
- Supplemental file 2 -
Fig. S2 (Importance of NCOA4 in maintaining mitochondrial function)
PDF, 5.0M
- Supplemental file 3 -
Fig. S3 (Autophagy partially contributes to ferritin complex degradation under iron-deprived conditions)
PDF, 1.4M
- Supplemental file 4 -
Legends to Fig. S1 to S3
PDF, 77K
- Supplemental file 1 -
