EKLF knockdown in normal human CD34⁺ progenitors does not induce megakaryocytic gene expression in late differentiated erythrocytic cells. Human cord blood CD34⁺ cells were amplified for 3 days in medium favoring erythrocytic progenitor amplification, infected with either Eklf shRNA (■) or control SCR shRNA lentivirus (□), and grown for further 3 days in medium favoring terminal erythroid differentiation as described in “Lentivirus production and cell infection.” (A) Western blot analysis of EKLF performed on the whole GFP⁺ infected cell population. (B) Quantitative RT-PCR analyses of erythrocytic (Eklf, Gpa, and β-globin) and megakaryocytic (GpIIIa and GpIX) mRNA levels in total GFP⁺ infected cells (lanes 1-2) and in purified late erythrocytic GPA⁺CD36⁺GFP⁺ infected cells (lanes 3-4). Results are expressed as relative levels (normalized to Hprt mRNA) after standardization to the level determined in the whole cell population infected with control lentivirus (lane 1). Note that the levels of GpIIIa and GpIX mRNA observed in the whole cell population (lanes 1,2) were at least 10-fold lower than that observed in the whole cell population obtained in the culture conditions favoring both erythrocytic and megakaryocytic differentiation in Figure 4. Error bars represent SD.