Rescue of KLF1 and LMO2 expression in Myb-silenced CD34⁺ cells. (A) Flowchart reporting the experiment timing (expressed in days) after CB-derived CD34⁺ stem/progenitor cells purification (in the upper part) and after the last of 3 nucleofection cycles (reported as postnucleofection on the lower part). (B) qRT-PCR analysis of (i) c-myb, (ii) KLF1, and (iii) LMO2 expression 48 hours after nucleofection. For each transcript, data are reported as Relative Quantity (RQ) of the mRNA in LXIDN MYBsiRNA (transduced with the empty retroviral vector and transfected with the c-myb siRNA), LKLF1IDN NegCTR, LLMO2IDN NegCTR (transduced with the retroviral vector for the overexpression of KLF1 and LMO2, respectively, and transfected with the nontargeting siRNA), LKLF1IDN MYBsiRNA and LLMO2IDN MYBsiRNA (transduced with the retroviral vector for the overexpression of KLF1 and LMO2, respectively, and transfected with the c-myb siRNA) respect to the LXIDN NegCTR sample (transduced with the empty retroviral vector and transfected with the nontargeting siRNA), which was set as calibrator. (iv) Table summarizing c-myb, KLF1, and LMO2 expression data (mean ± 2 SEM; n = 3). Error bars in the graphs represent SD. *P ≤ .05 and **P ≤ .01 vs LXIDN NegCTR sample. (C) Flow cytometric analysis of GPA and CD41 markers at day 7 after nucleofection after liquid culture with a multilineage cocktail of cytokines (i,ii) or with a cocktail of EPO and SCF, supporting the erythroid differentiation (iii-iv). Flow cytometry data (expressed as percentages of GPA- and CD41-positive cells) are reported in the graphs (i) and (iii) as mean ± SD and are summarized in the tables in panels ii and iv, respectively, as mean ± 2SEM. Results come from 3 independent experiments. *P ≤ .05 vs LXIDN MYBsiRNA sample. (D) Morphological analysis of May-Grunwald-Giemsa–stained cytospins after both liquid multilineage (day 18, i-iv) (C) and liquid unilineage erythroid (day 13, vi-x) culture post-CD34⁺ cells purification. The images were captured by the microscope Axioskop 40 microscope by means of AxioCam HRc Digital Camera and Axiovision software 3.1 (all Carl Zeiss MicroImaging Inc). The images were then processed with Adobe Photoshop 7.0 software. Original magnification ×630. n = number of experiments.