shRNA-mediated Ubap2l downregulation results in reduced progenitor and HSC activity. (A) Schematic representation of the shRNA-mediated gene knockdown approach. (B) Ubap2l knockdown levels in Lin⁻SCA1⁺ cells infected with 2 different shRNAs targeting Ubap2l (sh1 and sh2) as determined by qRT-PCR. The values for Ubap2l mRNA levels are expressed relative to Tbp and Hprt. The average of 2 independent experiments is presented with the standard error of the mean. The values for shLuc were set to 1. *Statistically significant differences (shLuc vs sh1: P = .0060, shLuc vs sh2: P = .0011). (C) CFC content of Lin⁻SCA1⁺ cells infected with sh2 determined by morphological analysis at day 7 of the culture. Colonies were grown in the presence of puromycin. Representative of 2 independent experiments performed in duplicate. (D) Reconstitution activity of Lin⁻SCA1⁺ cells infected with the different Ubap2l shRNAs (see panel B) as assessed by the percentage of GFP⁺ cells in the peripheral blood of the recipients 4, 8, and 12 weeks posttransplantation. The average of 2 independent experiments (n = 6-9 mice per condition) is shown with the standard error of the mean. Results for sh1 and sh2 were combined. *Statistically significant differences (4 weeks: P = .0154, 8 weeks: P = .0006, 12 weeks: P = .0017). (E) GFP levels in BM of mice transplanted with Lin⁻SCA1⁺ cells expressing sh1 and sh2 16 weeks posttransplantation. The average of 2 independent experiments is presented with the standard error of the mean. *Statistically significant differences (shLuc vs sh1: P = .0210, shLuc vs sh2: P = .0053). (F) Effect of sh1 and sh2 on the repopulation activity of FLA2 leukemia cells as determined by the percentage of GFP⁺ cells in recipients’ BM when signs of leukemia appearance was noted. Representative of 2 independent experiments with the mean of 5 mice per condition shown with the standard error of the mean. *Statistically significant differences (shLuc vs sh1: P = .0021, shLuc vs sh2: P = .0070).