Knockdown of Sf3b1 compromises proliferative capacity of HSCs. (A) Knockdown efficiencies of shRNAs against Sf3b1. WT and Sf3b1^+/− LSK cells were transduced with the indicated shRNAs and cultured in the presence of SCF and TPO. At day 3 postinfection, GFP⁺Lin⁻c-Kit⁺ cells were purified by cell sorting, and the levels of Sf3b1 were analyzed by quantitative RT-PCR analysis. mRNA levels were normalized to Gapdh expression. Expression levels relative to that in the control cells transduced with an shRNA against Luciferase are shown as the mean ± SD for triplicate analyses. (B) Growth of WT and Sf3b1^+/− CD34⁻LSK HSCs upon knockdown of Sf3b1 in culture. One hundred WT and Sf3b1^+/− HSCs were transduced with the indicated shRNAs and cultured in the presence of SCF and TPO. Data are shown as mean ± SD (n = 5). The transduction efficiency was ∼80% as detected by GFP expression on flow cytometry. (C) Reconstitution capacity of WT and Sf3b1^+/− CD34⁻LSK HSCs upon knockdown of Sf3b1 in vivo. WT and Sf3b1^+/− HSCs were transduced with the indicated shRNAs as in (B), and 100 transduced HSCs were transplanted into lethally irradiated recipient (CD45.1⁺) mice together with 2 × 10⁵ CD45.1⁺ competitor BM cells. Data are shown as mean ± SD (n = 5). Chimerism of donor-derived CD45.2⁺ cells (left panel) and CD45.2⁺GFP⁺ transduced cells (right panel) in the PB at 4 months posttransplantation are shown as mean ± SD (n = 5). *P < .02; **P < .01; ***P < .001.