Journal: Nature medicine

Article Title: Mitochondrial iron chelation ameliorates cigarette-smoke induced bronchitis and emphysema in mice

doi: 10.1038/nm.4021

Figure Lengend Snippet: IRP2-associated mitochondrial iron loading and CS. ( a ) Representative Perls’ stained lung sections (n=3 mice per group) (left) ( n = 2 technical replicates) with quantification (middle) ( n = 10 images per mouse, n = 3 per group) and non-heme iron levels (right, n = 4 mice per group, n = 4 technical replicates) in WT mice exposed to RA or CS (1–6 months). Scale bars, 50 μm. Arrows indicate staining. n.c.; negative control. ( b ) Representative immunoblot (n=3 experiments) (left) with quantification (right) of transferrin and ferritin expression ( n = 5 per group, n = 2 technical replicates), ( c ) total non-heme iron (left, n = 5 mice per group , n = 4 technical replicates) and fold change mitochondrial (RA n = 11; CS 1 month n = 6, 4 months n = 8, 6 months n = 9) and cytosolic non-heme (RA n = 6; CS 1 month n = 11, 4 months n = 3, 6 months n = 5) iron (right) in WT mouse lungs exposed to RA or CS, n = 2 technical replicates. ( d ) Fold change non-heme iron (left, WT and Irp2 −/− ; RA n = 13, CS 1 and 6 months n =5, CS 4 months n =3) and heme-iron (right , WT and Irp2 −/− ; RA n = 10, CS 1–6 months n =5) in mitochondrial fractions from WT mouse lungs exposed to RA or CS, n = 2 technical replicates. ( e ) Mitoferrin 2 (WT and Irp2 −/− ; RA n = 8; CS 1 month n = 3, 4 months n = 6), ( f ) frataxin expression (WT RA n = 5; CS n = 3; Irp2 −/− RA n = 6; CS n = 6) in whole lung of mice exposed to RA or CS. ( g ) Mitochondrial non-heme (left , n = 4 per group), mitochondrial heme iron (right , n = 4 per group), ( h ) 3 hour 99m Tc-SC clearance (left) and IL-6 protein concentrations (right , n = 4 per group) in the lungs of WT and Fxn ki/ko mice exposed to RA or CS (1 month). ( i ) Schematic of mitochondrial iron loading regulated by Irp2. All data are mean ± s.e.m. * P < 0.05. ** P < 0.01, *** P < 0.005 by one-way ANOVA followed by Bonferroni correction. # P < 0.05 by student’s unpaired t -test.

Article Snippet: Immunoblot analyses were performed in whole lung homogenates or mitochondrial-enriched fractions isolated from the lungs of mice using standard immunoblotting techniques with the following antibodies: Irp2 (1:1000 7H6: sc-33682, 1:1000 Santa Cruz, NB100-1798, Novus Biologicals and Irp2 antibody from Tracey Rouault at a 1:1000 dilution), Irp1 (1:1000 NBP1-19412, aconitase 1 Antibody, Novus Biologicals), LC3B (1:2000 L7543, Sigma Aldrich), Atg 7 (1:2000 APG7 (H-300): sc-33211, Santa Cruz), transferrin receptor 1 (1:1000 CD71 (H-300): sc-9099, Santa Cruz), actin (1:10,000 A00158, Sigma Aldrich), electron transport chain components including COX MTCO1 (1:500 MitoProfile® Total OXPHOS antibody, ab110413, Abcam), ferritin (1:1000 H-53: sc-25617, Santa Cruz), tom20 (1:2000 FL-145, sc-11415, Santa Cruz), frataxin (1:1000 H-155: sc-25820, Santa Cruz), mitoferrin 2 (1:1000 P-12: sc-138430, Santa Cruz), cleaved- caspase 3 (1:500 Asp175, Cell Signaling technologies), Bcl2 (1:1000 sc-7382, Santa Cruz), FBXL5 (1:1000 N0039, Neoclone Biotechnology) COX4I2 (1:1000 H00084701-M01 Abnova) and HO-1 (1:1000 sc-1797, Santa Cruz).

Techniques: Staining, Negative Control, Western Blot, Expressing