let-7a/f inhibition induced HIC2 levels in adult primary human erythroblasts. (A) Schematic of the CD34⁺ in vitro expansion and erythroid differentiation culture system. (B) The expression levels of let-7a-5p and let-7f-5p measured by miRNA qRT-PCR upon let-7a/f inhibition in primary human adult erythroblasts. Results are normalized to miRNAs miR-16/103a/191 and shown as mean ± SD (2 independent donors with 2 technical replicates each). Other let-7 family members are shown in supplemental Figure 6. (C) HBB, HBG, percentage of HBG, HIC2 and BCL11A mature mRNA, and primary transcript measured by qRT-PCR in let-7a/f inhibited adult human primary erythroblasts. Results are normalized to AHSP and shown as mean ± SD (n = 3 to 4 independent donors). (D) Sample flow cytometry with intracellular HBG staining in primary human erythroblasts. (E) Quantification of HBG–containing F-cells by flow cytometry. Results are shown as mean ± SD (n = 3 independent donors with 2 technical replicates for each donor). (F) Western blot with indicated antibodies of extracts from let-7a/f inhibited primary human adult erythroblasts. (G) Scatterplot of the RNA-seq results in primary human adult erythroblasts. (H) Schematic showing high let-7 levels in adult primary erythroid cells repressing HIC2; depletion of let-7 using a tough decoy leads to higher HIC2 levels and resulting decrease in BCL11A. FPKM, fragments per kilobase per million.