Suppression of FANCL expression in human CD34⁺ cord blood stem and progenitor cells leads to diminished β-catenin activity and multilineage progenitor expansion. (A) Approximately 3000 to 6000 CD34⁺ cord blood stem and progenitor cells were transduced with scrambled (Scr) or FANCL shRNA and the LEF-TCF-eGFP reporter. Cells were analyzed for eGFP expression after 3 days of puromycin selection (7 days total in culture). Remaining eGFP-positive cells were calculated by multiplying the starting CD34⁺ cells by the proportion of viable cells and the proportion of eGFP-positive cells. Each data point is expressed relative to the Scr condition. Shown are from 3 independent experiments (except FANCL shRNA construct B) with replicates plotted separately on the graph. The average of the 3 experiments is shown as a horizontal line within the graph. (B) Methylcellulose colony-forming assays were carried out as described. Colony count and type were scored; CFUs, GEMM colonies, and GM colonies. Summarized data are shown for 4 independent experiments (mean ± SEM). There were very few erythroid burst-forming units in these experiments. *Not statistically significant; **P < .04, Student t test. For these studies, transduced CD34⁺ cells were selected for puromycin resistance. In the colony-forming assays, unselected (no puromycin) cells were also grown to control for any potential effects of viral transduction on cell viability and colony formation that are independent of FANCL suppression.