Essential role of GATA-1 downstream of IL-4 during moDC differentiation. (A) Peripheral blood monocytes were stimulated with GM-CSF plus IL-4 or GM-CSF alone for 6 days. Fresh monocytes or day 6–generated cells were analyzed for GATA-1 expression by using Western blot analysis. K562 cells are compared. Data are representative of 4 independent donors. (B) Silencing of GATA-1. Western blot of GATA-1 and actin expression of not infected, nonsilencing or GATA-1 shRNA infected K562 cells (top) and moDCs (bottom) at day 7 of differentiation. GFP⁺ cells were purified before analysis. (C) Monocytes were transduced with a lentiviral vector encoding nonsilencing or GATA-1 shRNA sequences (ie, shGATA-1-GFP or control). A simplified graphical representation of the vector backbone is shown. Infected monocytes were induced by GM-CSF/IL-4 to differentiate to moDCs. Gated GFP⁺ cells were analyzed by FACS for the indicated markers. FACS blots are representative of 2 independent experiments. Cell cultures were initiated with 10⁶ monocytes, and cell recovery at day 7 was greater than 70%.