EKLF knockdown enhances the megakaryocytic differentiation output of human CD34⁺ progenitors and decreases their expression of the known erythrocytic target gene Gpa. Human cord blood CD34⁺ cells were amplified for 3 days in E/MK medium, infected for 3 hours with either Eklf shRNA or control SCR shRNA lentivirus, and grown for further 7 days in E/MK medium as described in “Lentivirus production and cell infection.” Figure shows the analysis of GFP-positive cells performed 7 days after infection (typical results from 3 independent experiments). (A) Western blot analysis showing the drastic reduction of EKLF protein level in purified GFP⁺ cells infected with Eklf shRNA lentivirus (lane 2) compared with control shRNA lentivirus (lane 1). HSC70 protein is shown as loading control. (B) FACS analysis of GPA expression in GFP⁺ cells. Histograms show that most of the GFP⁺ cells infected with Eklf shRNA lentivirus still express GPA (black line, gate M1) but at lower level than cells infected with control shRNA lentivirus (filled histogram, gate M1). Control isotype histogram is shown as dotted line. The percentages of GPA-positive cells as well as the mean geometric fluorescence of GPA (in brackets) for each population are indicated. (C) Typical FACS analysis showing a 2-fold increase in the percentage of CD41⁺CD42⁺ differentiated megakaryocytic cells among GFP⁺ cells infected with the Eklf shRNA lentivirus compared with that infected with the control shRNA lentivirus (left panel). Numbers on plot are the percentages of total cells in the gate. Histogram (right panel) shows the mean and SD of the fold increase in megakaryocytic cells obtained in 3 independent experiments (P value of .05 in paired Student test). (D) Comparison of erythrocytic (lanes 1,2) and megakaryocytic (lanes 3-5) gene mRNA levels in GFP⁺ cells infected with Eklf or control shRNA lentivirus. Relative levels of each mRNA have been determined by quantitative RT-PCR using Hprt as a normalization reference. Final results are expressed as fold changes induced by EKLF knockdown (shEKLF/shSCR ratios; means and SD from 3 independent experiments).