Gfi-1B knockdown impairs proliferation in primary human immature progenitor cells. (A) Experimental protocol to test the effects of Gfi-1B knockdown in immature primary human progenitors. CD34⁺ cells were purified from cord blood and amplified for 24 hours in the presence of IL-3, SCF, TPO, and FL and then infected with lentiviruses carrying shControl or shGfi-1B. Forty-eight hours after infection (D4), CD34⁺GFP⁺ cells were sorted and cultured in liquid culture in the presence of IL-3, IL-6, SCF, and EPO to induce the erythroid (E0-E7) or SCF and TPO for megakaryocytic differentiation (T0-T10). (B) Proliferation of shControl or shGfi-1B CD34⁺ cells. After CD34⁺GFP⁺ sorting, cells were cultured in the presence of SCF, FL, IL-3, and TPO and counted every day (corresponding to D5-D9 in the experimental protocol described in panel A) in the presence of Trypan blue. Error bars represent SD; n = 4 from different samples. (C-D) Flow cytometry histograms after annexin V and 7-AAD staining (C) and proportion of the cells in different phase of the cell cycle after 7-AAD and propidium iodide staining (D). Infected shControl and shGfi-1B CD34⁺ cells were analyzed at D5 and D7 or D9. (E) ShGfi-1B efficiency during erythroid and megakaryocytic differentiation. Cell lysates (noninfected cells [NI] and shControl or shGfi-1B for infected cells) were prepared at different times after induction of erythroid (E3 and E6) or megakaryocytic differentiation (T3 and T4) and analyzed by Western blotting with antibodies against Gfi-1B, Gfi-1, p21^cip/waf1, or β-actin (as control). The membrane was highly exposed after hybridization with Gfi-1B antibody to determine the efficiency of the Gfi-1B shRNA.