Prolonged treatment of PV mice with minihepcidin can cause iron-restricted erythropoiesis. PV mice (Jak2^V617F/+ VAV-Cre double transgenic) generated by BM transplantation (as seen before) start receiving M009 or vehicle by SC injections 4 weeks post-BM transplant (at a dose of 10, 15, or 20 mg/kg) twice a week for 6 weeks. Hematological parameters such as HCT (A), Hb (B), and RBC (C) were normalized in a dose-dependent fashion. However, this prolonged treatment was also associated with the development of iron-restricted erythropoiesis. This was indicated by increased splenomegaly (D) and erythroid maturation block shown by flow cytometry studies (E). Prussian blue staining on liver and spleen sections highlights increased iron deposition in the spleen, but not changes in liver iron content (F). 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. Results represent mean ± SD; ****P < .0001, ***P < .001, **P < .01, and *P < .05. Analysis was performed using one-way ANOVA with Tukey multiple comparison adjustment.