SLFN14^K208N/+mice exhibit splenomegaly and extramedullary erythropoiesis. (Ai) Representative images of spleens from SLFN14^K208N/+ and SLFN14^+/+ mice. (Aii) Normalized spleen weight. Spleen weight/body weight (mg/g) from 8 or 9 mice per genotype. (Bi) Representative images of H&E-stained spleen sections from SLFN14^K208N/+ and wild-type controls. Arrowheads indicate MKs. Scale bars, 50 µm. (Bii) Quantification of MK number per field of view. n = 3 mice per genotype, 10 or 11 fields of view per tissue sample. Analysis was conducted blind. (C) Quantification of MKs in spleen. (Ci) MK staining: MKs were identified by CD41 (αIIb) allophycocyanin (APC) and CD42 (GPIb) fluorescein isothiocyanate (FITC) double staining. (Cii) Proportion of MKs in spleen flow cytometry. (D) Quantification of erythroid progenitors in spleen. (Di) ProE staining: ProEs were identified as double-positive CD71 (transferrin receptor 1) phycoerythrin (PE) and Ter119 FITC cells (ProE gate). (Dii) Quantification of ProEs, increased Ter119⁺ cell population in SLFN14^K208N/+ mice, and profile of Ter119^hi cells by EryA, EryB, and EryC gates. (E) Quantification of MK-EB–primed MEPs in the spleen: MEPs were identified as a small population positive for CD71 (transferrin receptor 1) PE and CD41 (αIIb) APC (MEP). (Ei) Flow cytometry plots show a slight, but insignificant, increase in MEP cell numbers in SLFN14 K208N mice compared with wild-type. (Eii) MEP quantification. All spleen flow cytometry data and quantification are representative of 4 or 5 mice per genotype/staining condition. (F) Representative images of hemosiderin deposits in spleen sections of wild-type and SLFN14^K208N/+ mice highlighted by Perls Prussian blue staining. Scale bars, 50 µm. n = 3 mice per genotype. *P < .05, **P < .01, Student t test.