Inhibition of erythroid differentiation of MEL cells by knockdown of the Atf4 expression. (A) Knockdown of Atf4 expression in MEL cells by siRNAs. (B) Decreased expression of β-globin major mRNA in Atf4 knockdown cells. Data are presented as mean ± SD (n = 3, 3 separate transfections were performed). (C-D) Inhibition of erythroid differentiation in Atf4 knockdown cells. At 5 days after siRNA transfection, differentiating cells were stained with Giemsa and benzidine. Representative cells at different stages of differentiation stained are shown in panel C. Cell images were obtained with 40× objectives as describe for Figure 3B. (D) Percentages of cells at the basophilic erythroblast (Baso), polychromatic erythroblast (Poly), and orthochromatic erythroblast (Ortho) stages as well as benzidine-positive cells were determined by scoring the Giemsa-benzidine stained cells.