MAZ knockdown impairs erythropoiesis. (A) Flow cytometry analysis of CD71 and glycophorin (GPA) expression at day 15 of representative primary erythroblast differentiation cultures either untransduced (UT) or transduced with lentivirus expressing scramble shRNA (Scr), MAZ shRNA 699, or MAZ shRNA 703. (B) Quantitation of flow cytometry in A. Shown is the mean and standard deviation from 2 independent differentiation cultures. Burst-forming unit-erythroid (BFU-E) are CD71⁻/GPA⁻, and colony-forming unit-erythroid (CFU-E)/Proerythroblasts (Pro-E) are CD71⁺/GPA^lo/int and intermediate erythroblasts (Int. ery) are CD71⁺/GPA⁺. (C) Cell pellets from primary cultures at d15 of differentiation. Cultures with the MAZ shRNA 699 and 703 are pale compared with Scr control, indicating less extensive hemoglobinization. (D) Real-time RT-PCR analysis of expression of MAZ, HBA and HBB after 15 days of primary erythroid differentiation cultures. Shown is the mean and standard deviation from 2 independent differentiation cultures. For each gene, expression (relative to the PABPC1 gene) is normalized to the mean value observed with scramble shRNA. (E) Western blot analysis of the expression of MAZ, HBA, and HBB after 15 days in 2 independent primary erythroid differentiation cultures. β-actin was used as loading control. (F) Densitometry analysis of the western blots shown in E. For each protein, expression (relative to β-actin) is normalized to the mean value observed with scramble shRNA. (G) Variants around the MAZ locus significantly associated with changes in clinical erythroid traits (adjusted P value < .01). APC, allophycocyanin; PE, phycoerythrin; UTR, untranslated region.