SATB1 family protein and primary human adult erythroid progenitor cells. (A) Human primary hematopoietic progenitor cells were cultured in the presence of erythropoietin to stimulate erythropoiesis; harvested at days 0, 5, 8, 10, and 12, as indicated; and subjected to Western blot analysis using anti-SATB1 antibody (SATB). K562 cell lysate was used as a control and β-actin (βac) was used as a loading control. (B) Gene expression was determined for ϵ-, γ-, and β-globin in corresponding cultures of erythroid progenitor cells following erythropoietin stimulation at days indicated. Results were normalized to β-actin gene expression. (C) An SATB1 expression vector (2.5 μg DNA) was transfected using human CD34⁺ cell-specific Nucleofector solution (Amaxa, Köln, Germany) and electroporation (Amaxa program U-8) into primary erythroid progenitor cultures after 5 days of stimulation with erythropoietin (1 U/mL). Cells were harvested at day 8 and ϵ-, γ-, and β-globin gene expression was determined in SATB1-overexpressing cells (▪) compared with control (□). (D) ChIP DNA from primary erythroid progenitor cells with (▪) and without (□) SATB1 overexpression was isolated using antibodies specific for acetylated histone H3 (H3) and acetylated histone H4 (H4). Quantitative real-time PCR analysis using primers and TaqMan probes specific for the ϵ-globin and γ-globin promoters indicated the amount of associated acetylated histones. (E) Antisense oligonucleotide was used to down-regulate SATB1 family protein expression compared with the sense oligonucleotide shown in the Western blot using anti-SATB1 antibody and β-actin as a control. (I and II) Two independent primary erythroid progenitor cell cultures were treated with antisense oligonucleotide and ϵ- and γ-globin gene expression was measured. The results are given as fold change relative to the sense oligonucleotide control. Error bars represent SD from 3 independent measurements.