SPOP depletion increases γ-globin in primary erythroid CD34+cells. (A) Schematic diagram of experimental design. (B) Representative HbF flow cytometry of CD34+ erythroid cells at day 14 of differentiation. (C) Western blot (WB) analysis with indicated antibodies for differentiated CD34+ cells infected with scrambled or SPOP shRNAs virus. (D) High-performance liquid chromatography (HPLC) analysis of cells expressing indicated shRNAs at days 15 to 16 of differentiation. Arrow indicates HbF. (E) γ-Globin mRNA was measured by RT-qPCR; data are plotted as percentage of γ-globin over γ-globin + β-globin. Data are mean ± SD of 3 independent donors. (F) RNA-seq analysis for CD34+ cells at day 14 of differentiation. HBG1/2 indicates the combined levels of HBG1 and HBG2. The r value denotes Pearson correlation coefficient. Log2 FPKM values were averaged from 2 independent donors. EPO, erythropoietin; HbA, adult hemoglobin, HSC, hematopoietic stem cell; IL-3, interleukin-3; SCF, stem cell factor. **P < .05, unpaired Student t tests.