DEK is necessary for normal granulocytic differentiation of CD34⁺ cells. BM-derived CD34⁺ cells were transduced with lentiviral particles carrying shRNA sequences that targeted either DEK (shDEK1 or shDEK2) or an insertless construct as a negative control (KH1), or shRNA sequences targeting DEK #13104 and 04 or a nontargeting sequence (NT) as a negative control (Mission pLKO.1-puro UbC-TurboGFP vector, SHC014; Sigma-Aldrich; supplemental Table 3). After 3 days, separate aliquots of transduced cell cultures were assayed for either DEK protein expression or differentiation capacity. Cells were enriched for a GFP^high population and then probed by Western blot using 2 different anti-DEK Abs to determine the efficiency of DEK depletion through use of KH1 (A) or Mission (B) constructs. TATA-binding protein was used as a loading control. GFP^high enriched populations (1000 cells) from KH1 (C) and Mission (D) transduction constructs were plated in triplicate in MethoCult SF complete methylcellulose medium supplemented with cytokines the rhSCF, rhGM-CSF, rhG-CSF, rhIL-3, and rhIL-6. Colonies were scored after 14 days of incubation according to morphologic criteria: CFU-GM, CFU-GMM, CFU-G, or CFU-M. Each bar represents the mean of triplicate measurements ± SEM. (E-H) Transduced cells were incubated in the presence of rhG-CSF (20 ng/mL) for up to 10 days to induce granulocytic differentiation. Expression of the myeloid surface markers CD11b (E-F) and CD15 (G-H) were assessed after 7 or 10 days as indicated. Each bar represents the mean of 2 independent experiments ± SEM. Expression of CEBPE (I) and GCSFR3 (J) mRNA in GFP^high populations carrying KH1 transduction constructs after G-CSF treatment for 0 and 4 days. Expression levels are normalized against mRNA expression of 36B4. Error bars indicate the SD resulting from 2 independent experiments performed in triplicate. ***P < .001; **P < .01; *P < .05.