Fig. 3. BCL11A acts as a silencer of γ-globin gene expression, based on modulation of BCL11A levels. BCL11A acts as a silencer of γ-globin gene expression,

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Fig. 3. BCL11A acts as a silencer of γ-globin gene expression, based on modulation of BCL11A levels. BCL11A acts as a silencer of γ-globin gene expression, based on modulation of BCL11A levels. (A) siRNA-mediated knockdown of BCL11A results in elevations of γ-globin mRNA levels (as a percentage of total β-like globin gene expression) in human erythroid progenitor cells at day 7 of differentiation in comparison with NT control siRNAs. (B) Microarray profiling of these cells using the Affymetrix U133 Plus 2.0 array reveals that there is close similarity in the expression profile of NT and BCL11A siRNA-treated cells (r2 = 0.9901). Microarray data processing and filtering were performed as described previously (27) and in (11). (C) Lentiviral-mediated shRNA delivery to human erythroid progenitors results in robust knockdown of BCL11A protein. Control samples were infected with lentivirus prepared from the backbone pLKO.1ps vector. (D) At day 6 of differentiation, the cells appear to be morphologically indistinguishable; this is also the case at other stages of differentiation. (E) The shRNA-mediated knockdown of BCL11A results in robust induction of γ-globin mRNA level on day 7 of differentiation (***P < 10–5 in comparison with control). (F) Hemolysates prepared from cells on day 12 of differentiation show the presence of mature HbF by hemoglobin HPLC. The HbF peaks are labeled with an arrow in each chromatogram, with the first peak corresponding to acetylated HbF (28) and the second unmodified HbF. All results are means ± SEM. Statistical significance was calculated using the Student's t test. Vijay G. Sankaran et al. Science 2008;322:1839-1842 American Association for the Advancement of Science