Fig 2: MicroRNA29B (MIR29B) mediates HBG reactivation via MYB gene silencing in normal human erythroid progenitors. (A) Wright-Giemsa-stained images of human erythroid progenitors (n = 3) undergoing differentiation at day 3, day 7, and day 12 in vitro. Quantitative RT-qPCR analysis of (B) MYB mRNA relative to β-actin, (C) MIR29B relative to RNU48, and (D) HBG-to-HBB mRNA ratio as a function of HBG/HBG + HBB, (E) Representative histograms from flow cytometry analysis of the percentage of HbF-positive cells following transfection of MIR29B alone or in combination with MYB into primary erythroid progenitors (n = 3) stained with FITC anti-HbF antibody. (F) Bar graphs showing quantification of the ratio of HbF-to-HbA in erythroid progenitors (n = 3) transfected with MIR29B alone or in combination with MYB. *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001 is statistically significant.

Fig 3: MicroRNA29B (MIR29B) mediates HBG reactivation via inhibiting MYB in KU812 cells. (A) Western blot analysis of MYB and HbF protein expression relative to β-actin protein in KU812 cells transfected with increasing concentrations of MYB plasmid DNA (μg). Fold change of (B) MYB mRNA relative to β-actin, (C) MIR29B expression relative to RNU48, and (D) HBG-to-HBB mRNA ratio as a function of HBG/HBG + HBB mRNA expression in KU812 cells following transfection for 48 h with MIR29B mimic (50 or 100 nm) alone or in combination with MYB overexpression plasmid vector (2 μg). (E) Western blot of MYB, HbF, and HbA protein expression with β-actin internal control. (F) Bar graphs showing quantification of the ratio of HbF-to-HbA protein in KU812 cells transfected with MIR29B alone or in combination with MYB. *P < 0.05 **P < 0.005, ***P < 0.0005, and ****P < 0.0001 is statistically significant.