Figure 4.

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 ^99mTc-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.