Rescue of erythroid and megakaryocytic differentiation by knockdown of TGFBR3 in Gfi-1B–depleted CD34⁺ cells. (A) Experimental protocol to test the effects of TβRIII shRNA in Gfi-1B knockdown immature primary human progenitors. CD34⁺ cells were amplified for 24 hours in the presence of IL-3, SCF, TPO, and FL and then infected with lentiviral vectors carrying shControl or shGfi-1B or with both of them (shGfi-1B+shΤβRΙΙΙ). The day after the second transduction, cells were selected for puromycin resistance; and after 24 hours, GFP⁺ cells were sorted by FACS. Then, cells were either plated in semisolid medium to determine the number of erythroid or megakaryocytic progenitors or cultured in liquid culture in the presence of EPO and SCF. (B) TβRIII shRNA efficiency in K562 cells. K562 cells were transduced with lentiviral vectors containing the TβRIII shRNA and selected in the presence of puromycin. At 48 hours after the beginning of the selection, cell lysates were prepared and subjected to Western blot analysis with a TβRIII-specific antibody. (C-D) Erythroid (C) and megakaryocytic (D) colony formation in semisolid medium. As described in Figure 3A, puromycin-resistant GFP⁺-infected cells were plated in Methocult in the presence of EPO, IL-3, IL-6, and SCF or in Megacult in the presence of TPO, IL-6, and IL-3. Error bars represent SD. **P < .002; n = 2 with different samples. (E) Analysis of erythroid differentiation. GPA expression was analyzed by flow cytofluorometry 5 days after induction of erythroid differentiation of shControl-, shGfi-1B-shTβRIII–, or shGfi-1B+shTβRIII–transduced cells.