Down-modulation of BMI1 by lentiviral RNAi in human CB CD34⁺ cells impairs proliferation and reduces CFC and LTC-IC frequencies. (A) CB CD34⁺ cells were transduced with control (scrambled) scr-RNAi or BMI1-RNAi particles and sorted; mRNA was isolated; and BMI1 expression was analyzed by quantitative reverse transcription (RT)–PCR analysis. (B) As in panel A, but now total lysates were prepared and analyzed by Western blotting. (C) Transduced CB cells were grown in long-term cocultures on MS5 bone marrow stromal cells. Cultures were weekly analyzed, and the growth curve represents cumulative cell numbers during the culture period. A representative experiment of 3 independent experiments is shown. (D) Hematopoietic differentiation was analyzed by FACS on suspension cells from MS5 cocultures at week 1. (E) Suspension cells were harvested from MS5 cocultures, as described in panel C, and progenitor content was determined by CFC assays in methylcellulose. (F-G) Lentiviral transductions as in panel A, but now stem cell frequencies were determined in limiting dilution (F) or in bulk T25 flasks (G).