Gene silencing by lentiviral delivery of p53 siRNA. (A) Reduction of p53 mRNA expression by siRNA. CD34⁺ cells were infected and sorted for EGFP-positive cells. Expression of p53 mRNA was assessed by quantitative RT-PCR at the day of sorting (left panel) and after 5 weeks of liquid culture (right panel). The value for the control cells infected with pWPXL virus (▪) was set as 100% and compared with cells infected with the pWPXL-control-si virus (▦) and the pWPXL-p53si virus (□). Error bars indicate the SEM when triplicate measurements were done. (B) Sorted CD34⁺/GFP⁺ cells were cultured in methylcellulose and single colonies were picked after 14 days for detection of p53 mRNA. Note that ΔCT (cycles threshold) represents a binary logarithmic scale and higher numbers correspond to lower levels of p53 expression. Each dot represents the result obtained from one single colony. transd. indicates transduced. (C) Effects of etoposide on CFU-C formation. Sorted CD34⁺/EGFP⁺ cells were grown in methylcellulose in the presence (+) or absence (-) of etoposide (25 nM). Error bars indicate SEM of triplicate cultures. ^* indicates a statistically significant difference (P < .01). (D) Measurement of etoposide-induced apoptosis by flow cytometry. CD34⁺ cells infected with pWPXL-p53si or the empty vector were sorted for EGFP expression by FACS and cultured for 4 days in liquid culture. After exposure to 20 μM etoposide (Eto) for 6 hours, apoptosis was measured by staining with PE-coupled annexin V. The percentages of Annexin V/EGFP-double-positive cells are given. (E) p53 target gene expression in silenced versus control cells. p21 mRNA levels were determined by real-time PCR in sorted EGFP-positive cells after 10 days in liquid culture. Annotation as in panel A. (F) Silencing of p53 expression in sorted CD34⁺/GFP⁺ cells after 5 weeks of LTC-IC stroma-cell culture. Annotation as in panel A. (G) Silencing of p53 expression in LTC-IC-derived colonies. Annotation as in panel B.