Article Figures & Data
Figures
- FIG. 1.
Time-dependent inactivation of IRP1 and IRP2 by SNP and FAC. RAW 264.7 cells were either left untreated as control (lanes 1, 4, and 7) or exposed to 100 μM SNP (lanes 2, 5, and 8) or 30 μg of FAC/ml (lanes 3, 6, and 9) for the indicated time intervals. (A) Cytoplasmic extracts (25 μg) were analyzed for IRE-binding activity with a 32P-labeled IRE probe in the absence (top) or presence of 2% 2-mercaptoethanol (2-ME; bottom). The positions of the IRE/IRP1 and IRE/IRP2 complexes and of excess free probe are indicated by arrows. (B) The band intensities of three representative experiments were quantified by phosphorimaging and plotted relative to control (mean ± the standard deviation [SD]).
- FIG. 2.
Accelerated turnover of IRP2 by SNP and FAC. H1299 cells were plated for 24 h in tetracycline-free medium to express IRP2wt-HA. Tetracycline (2 μg/ml) was then added back to shut off the transcription of the IRP2wt-HA cDNA. (A) After 1 h, the cells were either left untreated (lanes 1 to 3) or exposed to 100 μM SNP (lanes 4 to 5), 100 μM K3[Fe(CN)6] (lanes 6 to 7), 30 μg of FAC/ml (lanes 8 to 9), or 100 μM DFO (lanes 10 to 11) for the indicated time intervals. (B) Likewise, the cells were either left untreated (lanes 1 to 3) or exposed to 100 μM KCN (lanes 4 to 5), 100 μM DFO (lane 6), 100 μM SNP (lane 7), or 30 μg of FAC/ml (lane 8). Cell lysates were subjected to Western blotting with HA (top) and β-actin (bottom) antibodies. The immunoreactive bands were quantified by densitometric scanning. The IRP2wt-HA/β-actin ratios are plotted (mean ± the SD) over time to illustrate the effects of the treatments on IRP2 decay.
- FIG. 3.
Pharmacological inhibition of IRP2 degradation by SNP and FAC. H1299 cells were plated for 24 h in tetracycline-free medium to express IRP2wt-HA. (A and B) Tetracycline (2 μg/ml) was added back to shut off the transcription of the IRP2wt-HA cDNA. After 1 h, the cells were either harvested (lanes 1), left untreated (lanes 2), or treated with 100 μM SNP (A) or 30 μg of FAC/ml (B) in the absence (lanes 3) or presence of 5 μM actinomycin D (ActD; lanes 4), 40 μM cycloheximide (CHX; lanes 5), or 100 μM DFO (lanes 6) for 8 h. Lysates were analyzed by Western blotting with HA (top) and β-actin (bottom) antibodies. (C) After an overnight pretreatment with 100 μM DFO, the cells were either left untreated (lane 1) or treated for 6 h with 100 μM SNP (lanes 3 to 4) or 30 μg of FAC/ml (lanes 5 to 6) in the absence or presence of 5 mM DMOG (lanes 2, 4, and 6). Lysates were analyzed by Western blotting with HA (top), HIF-1α (middle), and β-actin (bottom) antibodies. (D) The cells were either left untreated (lane 1) or treated for 12 h with 100 μM SNP (lanes 3 to 4) or 30 μg of FAC/ml (lanes 5 to 6) in the absence or presence of 2 mM succinyl acetone (SA; lanes 2, 4, and 6). Lysates were analyzed by Western blotting with HA (top) and β-actin (bottom) antibodies.
- FIG. 4.
IRP2 mutants bearing either C168S, C174S, and C178S substitutions or a deletion of the “73-aa domain” are sensitive to SNP-mediated degradation. H1299 cells were plated for 24 h in tetracycline-free media to express IRP2wt-HA (A), IRP23CS-HA (B), or IRP2Δ73-HA (C). The cells were either left untreated (lanes 1) or were treated for 12 h with 100 μM SNP (lanes 2 to 3) or 30 μg of FAC/ml (lanes 4 to 5) in the absence or presence of 20 μM MG132 (lanes 3 and 5). Lysates were analyzed by Western blotting with HA (top) and β-actin (bottom) antibodies.
- FIG. 5.
IRP2 undergoes proteasomal degradation in response to fresh and photodegraded SNP, but remains unaffected by GSNO. (A) H1299 cells were plated for 24 h in tetracycline-free medium to express IRP2wt-HA. The cells were either left untreated (lane 1) or were treated for 12 h with 100 μM fresh (lanes 3 to 4) or photodegraded (lanes 5 to 6) SNP or 100 μM GSNO (lanes 7 to 8) in the absence or presence of 20 μM MG132 (lanes 2, 4, 6, and 8). Lysates were analyzed by Western blotting with HA (top) and β-actin (bottom) antibodies. (B) Solutions of freshly prepared and photodegraded SNP were analyzed for the presence of nitrite by using Griess reagent. (C) H1299 cells were exposed to 1 mM fresh or photodegraded SNP for the indicated time intervals, and the nitrite levels in the culture supernatant were determined by using the Griess reagent.
- FIG. 6.
Fresh and photodegraded SNP, but not GSNO, promote an increase in the LIP. RAW 264.7 cells were either left untreated (ctrl) or were treated for 30 min with 30 μg of FAC/ml or with 100 μM hemin, fresh SNP, photodegraded SNP, K3[Fe(CN)6], or GSNO. Relative alterations of the LIP were registered with the calcein assay upon addition of isonicotinoyl-hydrazone salicyladehyde. Values correspond to triplicate samples (mean ± the SD).
