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Mechanisms of NCOA4-maintained murine erythropoiesis
to the levels after acute depletion (Figure 2E). This normal- ization is consistent with an increase in hepcidin levels (Figure 2F), increase in serum iron and resolution of ane- mia. However, Fpn levels were sustainably increased in
the duodenum as part of a compensatory response to iron deficiency and, in part, explaining the high iron in serum. It is to be expected that duodenal Fpn expression would decrease with a longer period of time as demonstrated in
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Figure 1. Acute systemic knockout of Ncoa4 impairs ferritinophagy and erythropoiesis. A murine model of Ncoa4 deficiency was generated using a tamoxifen- inducible Cre recombinase system (Ncoa4fl/fl;UBC-cre/ERT2). Ncoa4fl/fl and Ncoa4fl/fl;UBC-cre/ERT2 mice were administered tamoxifen to generate Ncoa4-deficient (Ncoa4rec) and control animals (Ncoa4fl/fl). (A) Genomic DNA isolated from spleens of Ncoa4fl/fl and Ncoa4fl/fl;UBC-cre/ERT2 yielded a polymerase chain reaction (PCR) product at 470 bp when using primers directed at the floxed allele (left panel, see Methods). This band was not detected in Ncoa4rec mice. Using primers directed at the recombined allele, a PCR product of 540 bp was detected for Ncoa4rec but not Ncoa4fl/fl (right panel). (B) Increased Fth1 protein levels in liver, spleen, kidney, brain but not pancreas or bone marrow from Ncoa4rec mice 11 days after initiation of tamoxifen administration. β-actin (Actb) served as a loading control. (C-E) Tissue iron levels in spleen (C), liver (D) and bone marrow (E) from Ncoa4rec in comparison to Ncoa4fl/fl mice [3-5 mice/group, error bars represent the standard error of mean (s.e.m.)] (F) Lower serum iron levels in Ncoa4rec mice (3 mice/group, error bars represent the s.e.m.). (G) Increased Fpn protein levels in spleen, liver, and duodenum from Ncoa4rec mice 11 days after initiation of tamoxifen administration. Actb served as a loading control. (H) Whole blood from adult (>12 weeks) Ncoa4fl/fl and Ncoa4rec mice 7 days after the last administration of tamoxifen was acquired by retro-orbital bleeding for complete blood count profiling (Ncoa4fl/fl, n = 12; Ncoa4rec, n = 11, error bars represent the s.e.m.). (I) Elevated erythropoietin levels in serum of Ncoa4rec mice (8 mice/group, error bars represent the s.e.m.). (J) Increased Hif- 2a protein levels in kidney from Ncoa4rec mice 11 days after initiation of tamoxifen administration. Actb served as a loading control. (K) No significant change in spleen size of Ncoa4fl/fl and Ncoa4rec male and female mice 11 days after the initiation of tamoxifen administration (4 males and 7 females/group). (L) Bach1, Hri, Eif2a and Eif2a-P protein levels in red blood cells of Ncoa4fl/fl mice and Ncoa4rec. Actb served as a loading control. For all panels, statistical comparison was performed using a two-tailed Student t-test: *P<0.05, **P<0.01, ***P<0.001. TAM: tamoxifen; Fth1: ferritin heavy chain 1; Fpn: ferroportin; RBC: red blood cell count; HCT: hematocrit; HGB: hemoglobin; MCH: mean corpuscular hemoglobin; CHr: reticulocyte hemoglobin content; MCV: mean corpuscular volume; RDW: red blood cell dis- tribution width; RETIC: reticulocyte count; PLT: platelet count; Epo: erythropoietin; b.w.: body weight.
haematologica | 2019; 104(7)
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