Erythroid differentiation is maintained overall with a combination of HRI knockdown and HbF pharmacologic treatments. (A) Representative flow cytometric analysis of erythroid differentiation in healthy CD34⁺ donors after shRNA scrambled control or HRI depletion with 2 independent shRNAs combined with either vehicle control or HbF pharmacologic induction (50 μM HU, 1 μM pomalidomide [Pom], or 0.125 μM UNC0638). Percentages denote frequency of parent population. (B) Quantification of flow cytometric analysis of erythroid differentiation by CD71⁺CD235a⁺ vs CD71⁻CD235a⁺ populations. Each symbol (circle, square, triangle) represents a biologically independent sample. (C) Representative Wright-Giemsa staining of erythroid cells. Cytospin images were captured at 10× resolution on an Olympus BX60 microscope with Infinity software. DMSO served as the pharmacologic vehicle control. Three independent biological replicates for all experiments. Error bars represent standard deviation. (D-F) RNA-Seq combinatorial analyses of DMSO-treated control shRNA samples vs DMSO-treated HRI shRNA #2 samples (D), DMSO-treated control shRNA samples vs pomalidomide-treated HRI shRNA #2 samples (E), and DMSO-treated control shRNA samples vs UNC0638-treated HRI shRNA #2 samples (F). Erythroid maturation upregulated genes (red) include (1) GATA1, (2) KEL, (3) ANK1, (4) KLF1, (5) FOXO3, (6) AHSP, (7) EPB42, (8) GYPA, (9) ALAS2, (10) SLC4A1 (BAND3), and (11) SLC25A37. Erythroid maturation downregulated genes (green) include (12) GATA2, (13) JUN, (14) MYC, (15) CD44, (16) MYB, (17) KIT, (18) CASP3, and (19) PCNA. Two independent biological replicates for RNA-Seq analysis.