Overexpression of SAR1 enhances HbF production in K562 and CD34⁺ cells. (A) Western blot analysis of SAR1 protein expression in K562 and CD34⁺ cells after 3 days of 100 µM HU treatment. CD34⁺ cells were treated with HU at day 5 of differentiation. (B) Western blot analysis of γ-globin expression in SAR1- or vector control–stably transfected K562 cells and SAR1- or vector control–transfected CD34⁺ cells. β-actin was used as a loading control. (C) SAR1- or vector control–stably transfected K562 cells were stained with PE-conjugated anti-HbF antibody, then subjected to flow cytometry to detect HbF-positive cells. Left panels, 2 representative flow cytometry results. Right panel, graphical representations of flow cytometry data for HbF protein expression in SAR1- or vector control–stably transfected K562 cells. *P < .05 vs vector control–transfected cells. Error bars represent SD of the mean of >3 independent experiments. (D) Real-time PCR analysis of γ-globin and SAR1 expression in CD34⁺ cells treated with HU, 5-AZA, or Ara-C for 72 hours on day 6 of differentiation. The vehicle-treated value was set to 1.0 and fold increase was calculated relative to expression in vehicle-treated cells after normalization to β-actin gene expression. *P < .05 vs vehicle-treated cells. Error bars represent SD of the mean of 3 independent experiments. 5-AZA, 5-azacytidine.