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Blood, Vol. 113, Issue 3, 679-687, January 15, 2009
Cell-autonomous and systemic context-dependent functions of iron regulatory protein 2 in mammalian iron metabolism
Blood Ferring-Appel et al.
113: 679
Supplemental materials for: Ferring-Appel et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 62.2 KB)
- Table S1. Plasma iron parameters in mice with total versus tissuespecific IRP2 deficiency (PDF, 418 KB) -
Plasma iron levels and transferrin saturation values were determined at 10 weeks of age in Ireb2+/+ versus Ireb2Δ∕Δ mice with total and constitutive IRP2 deficiency and in Ireb2VilCre, Ireb2AlfpCre, and Ireb2LysMCre mice with (+) or without (−) selective IRP2 ablation in intestinal epithelial cells, in hepatocytes, or in macrophages, respectively. Data are given as average ± SEM. The sample size (n) is indicated. TIBC, mean total iron binding capacity. TIBC = plasma Fe + unbound iron binding capacity, Transferrin saturation = (plasma Fe/TIBC) ×100. Data were compared between control and IRP2-deficient animals within each group; no statistically significant difference was observed (Student t-test).
- Table S2. Plasma ferritin levels in mice with total versus tissue-specific IRP2 deficiency (PDF, 296 KB) -
Plasma ferritin levels were determined at 10 weeks of age in Ireb2+/+ versus Ireb2Δ∕Δ mice with total and constitutive IRP2 deficiency and in Ireb2VilCre, Ireb2AlfpCre, and Ireb2LysMCre mice with (+) or without (−) selective IRP2 ablation in intestinal epithelial cells, in hepatocytes, or in macrophages, respectively. Data are given as average ± SEM. The sample size (n) is indicated. Data were compared between control and IRP2-deficient animals within each group; a p<1×10−2, b p<1×10−3, c p<1×10−9, d p<1×10−13, student t-test.
- Figure S1. Recombination efficiency in peritoneal macrophages from Ireb2LysMCre mice (JPG, 31.8 KB)
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The efficiency of Cre-mediated recombination in peritoneal macrophages from Ireb2LysMCre mice with (+) or without (−) Cre expression was analyzed by RNase protection assay for full length (corresponding to the functional floxed allele) versus truncated (corresponding to the null allele) IRP2 mRNA. A representative autoradiogram is presented, showing efficient, although not complete, ablation of full length IRP2 mRNA. β-actin was used as a loading control.
- Figure S2. IRE-binding activity of IRP1 in IRP2-deficient tissues (JPG, 63 KB)
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The IRE-binding activity of IRP1 was analyzed by electromobility shift assay (EMSA) in duodenal scrapings, in the liver, and in BMDM, respectively, of Ireb2VilCre, Ireb2AlfpCre, and Ireb2LysMCre animals expressing (+) or not (−) the Cre recombinase. (A) Representative EMSA autoradiographs are presented. IRP2/IRE complexes could not be detected in duodenal samples. Note that trace amounts of IRP2/IRE complexes could still be detected in BMDM of Ireb2LysMCre(+) mice. (B) The histograms (mean ± SD) show the IRE-binding activity of IRP1 quantified from a larger number of animals per group, as indicated (n). Within each group, the IRE-binding activity of IRP1 of littermates lacking Cre expression was set to 100%. A student t-test revealed no statistically significant variation of the IRE-binding activity of IRP1 in IRP2-deficient tissues.
- Figure S3. Perl’s staining for iron of spleenic tissues of mice with macrophage-specific versus total IRP2 deficiency (JPG, 142 KB)
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Splenic sections from Ireb2Δ∕Δ mice with total and constitutive IRP2 deficiency versus Ireb2+/+ littermates and from Ireb2LysMCre mice with (+) or without (−) Cre expression were subjected to Perl’s staining in order to detect iron in macrophages (blue coloration). While splenic macrophages of Ireb2Δ∕Δ animals are relatively iron-deficient compared to Ireb2+/+, Ireb2LysMCre(+) mice with selective ablation of IRP2 in macrophages display normal iron staining when compared to Ireb2LysMCre(−) littermates. Pictures were acquired using a 10× objective (NA 0.30). Counterstain: Nuclear Fast Red. RP: red pulp, WP: white pulp.
- Figure S4. Ferroportin immunostaining in the spleen of mice with macrophage-specific versus total IRP2 deficiency (JPG, 194 KB)
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Splenic sections from Ireb2Δ∕Δ mice with total and constitutive IRP2 deficiency versus Ireb2+/+ littermates and from Ireb2LysMCre mice with (+) or without (−) Cre expression were subjected to ferroportin (FPN) and F4/80 immunostaining. In agreement with a previous report4 and with our western-blot analysis (Fig. 4C), the FPN signal (green) is strongly decreased in the vast majority of red-pulp macrophages (labelled with F4/80, red) from Ireb2Δ∕Δ mice versus Ireb2+/+. By contrast, macrophages from Ireb2LysMCre(+) animals display no change in FPN expression. Pictures were acquired using a 40× oil-objective (NA 1.25). Scale bar: 50 µm.
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