The iron-restrictive effect of minihepcidin peptide M004 improved ineffective erythropoiesis in a mouse model of thalassemia intermedia. Administration of M004 (2.625 mg/kg) in 2-month-old Hbb^th3/+ animals twice a week for 6 weeks resulted in increased RBC count (A), improved anemia (as shown by increased Hb levels) (B), reduced splenomegaly (C), and reticulocyte count (D). Hemichrome formation was markedly reduced, as indicated by the reduced intensity of the α precipitates in animals treated with minihepcidin compared with control group (TAU gel) (E). ROS formation was also significantly reduced (F). To measure ROS levels, the erythroid compartment was at first analyzed by staining BM and spleen cells with CD44 and Ter119 antibodies that allowed the separation of erythroid cells into 5 distinct populations. The cells were then stained for ROS detection using the indicator CM-H2DCFDA. Here, we show a representative example of the ROS-positive cells (for control and treated groups) in the fraction that represents mature RBCs (F). RBC morphology (shown by Giemsa staining of peripheral blood smear) was markedly improved by M004 administration (G) and lifespan normalized (H). As a result of decreased erythroid iron uptake, total organ iron content was significantly reduced in liver (I) and kidney (J). Perls’ Prussian blue staining on liver, spleen, and kidney sections of animals receiving vehicle or M004 shows that parenchymal iron is reduced (liver, kidney) while retention of iron in the splenic macrophage is increased (K). Images were captured using a Leica DM4000B upright scope paired with a Spot RT/SE Slider camera (10×/numerical aperture 0.40 objective) and then acquired using the Spot 5.1 software. Perls’ Prussian blue staining in spleen sections revealed increased iron retention, but when organ weight was taken in consideration, the spleen iron concentration was statistically reduced in M004-treated vs vehicle-treated animals (L). Results are presented as mean ± SD; ***P < .001, **P < .01, and *P < .05, 2-tailed Mann-Whitney U test. WT data are displayed as a reference guide but are not included in the between-group test comparisons. st, standard.