Figure 5.

COX is pathogenic in experimental COPD. (a) Coa3 expression (left) in WT (n = 6) and Irp2^−/− (n = 5) mouse lungs, schematic of COX assembly (middle) and COX expression by immunoblot analysis (right) in lung tissue from individuals with COPD (n = 5) and controls (n = 5), n = 2 technical replicates. (b) Representative immunoblot (n=3 experiments) expression (left) of OXPHOS complexes I-V in mitochondrial-enriched fractions from WT and Irp2^−/− mouse lungs exposed to RA or CS (4 months), with quantification of Complex IV expression (right), n = 3 technical replicates. (c) Time course of COX activity (left) and total COX activity (4 months CS) in mitochondrial fractions of WT or Irp2^−/− mice exposed to RA or CS (n = 3 per group, n = 3 technical replicates). (d) Representative immunoblot (n=3 experiments) (left) as in (b), time course of COX activity (middle) and COX activity at 4 hours (right) in primary lung epithelial cells from WT or Irp2^−/− mice exposed to 20% CSE, n = 2 per group, n = 2 technical replicates. (e) 3 hour ^99mTc-SC clearance (left), total BALF leukocytes (middle), total protein levels (right), (f) BALF IL-33 (left, ELISA; WT RA n = 4; CS n = 5; Sco2^ki/ko RA n = 5, CS n = 4), BALF IL-6 (right, ELISA; WT RA n = 4; CS n = 5; Sco2^ki/ko RA n = 5, CS n = 4),) protein concentration, (g) total lung non-heme iron (n = 4 per group), (h) mitochondrial non-heme iron (left, n = 3 per group) and mitochondrial-heme iron (right, n =3 per group) levels in WT and Sco2^ki/ko mice exposed to RA or CS (1 month), (f, g) n = 3 technical replicates. (i) Schematic of the role of COX Irp2-associated mitochondrial iron loading. 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, ^##P < 0.01, ^###P < 0.005 by unpaired student’s t-test.