SCF suppresses COUP-TFII binding to the γ-globin promoter and knockdown of endogenous COUP-TFII induces γ-globin expression in erythroblasts. (A) Antibodies against COUP-TFII and RNA polymerase II (PoL II) were used to immunoprecipitate chromatin. Precipitated DNA was amplified and quantitated by real-time PCR with primers flanking the γ-globin gene promoter. Results are expressed as relative proportions of immunoprecipitated DNA (ratios of immunoprecipitated versus input DNA) normalized to the ratio obtained for the γ-globin promoter in unstimulated cells (arbitrarily set at 1). (B) Effect of different doses of silencing COUP-TFII mRNA or protein levels was evaluated by quantitative RT-PCR or immunoblot analysis on day 12 cultured cells after transiently transfected with either COUP-TFII or control non–targeting siRNA by Amaxa electroporation. (C) Quantitative RT-PCR analysis of γ-, β-globin mRNA levels, and the ratio of γ/(γ + β) globin percentages in negative control or knockdown COUP-TFII (different doses) cells. (D) Representative fields of Giemsa-stained erythroid cell at day 12 after knockdown of COUP-TFII at different doses. Results are shown as mean ± SD from 3 independent donors that were analyzed in separate experiments. *P < .05 versus negative control siRNA.