Angptl3 represses the expression of Ikaros to maintain stemness. (A) Angptl3-null HSCs had increased expression of Ikaros compared with WT HSCs. Lin⁻Sca-1⁺Kit⁺Flk2⁻CD34⁻ cells were collected from WT or Angptl3-null BM. Gene expression analyzed by real-time RT-PCR is shown. The gene expression in WT samples was normalized to 1. (*significantly different from WT values, P < .05, n = 7). (B) The real-time RT-PCR analysis of bone marrow Lin⁻Sca-1⁺Kit⁺Flk2⁻CD34⁻ cells cultured in SCF-containing serum-free medium, treated with or without 500 ng/mL Angptl3 for 24 hours. Gene expression in untreated samples was normalized to 1. (C) The real-time RT-PCR analysis of WT and Angptl3-null bone marrow Lin⁻Sca-1⁺Kit⁺Flk2⁻CD34⁻ cells cultured in serum-free STF medium, treated with or without 500 ng/mL Angptl3 for 8 days. Gene expression in Angptl3-null samples was normalized to 1. Results of averages of all real-time RT-PCR reactions are shown. (*P < .05, n = 3). (D) Forced expression of Ikaros increased Ikaros expression 2.5-fold. Mouse E16 fetal liver CD45.1 Lin⁻ cells were infected by retroviruses encoding GFP or Ikaros-IRES-GFP. The infection efficiency of Ikaros-IRES-GFP was approximately 20%. Three days later, 50 000 GFP⁺ cells were transplanted into lethally irradiated CD45.2 recipients. The expression of Ikaros in HSCs was quantitated by real-time RT-PCR 4 months after transplantation. (E) Overexpression of Ikaros decreased quiescence of HSCs. Cell cycle status of the donor GFP⁺Lin⁻Sca-1⁺Kit⁺ cells shown in panel D was analyzed by Hoechst/pyronin Y staining 4 months after transplantation (n = 6). (F) Mouse E16 fetal liver CD45.2 Lin⁻ cells were infected by retroviruses encoding GFP or Ikaros, and 1.7 × 10⁵ of these sorted GFP⁺ cells were cotransplanted with 10⁵ CD45.1 bone marrow competitors into lethally irradiated CD45.1 recipients for competitive repopulation analysis. Donor engraftment at indicated time after transplantation is shown (*P < .05, n = 9). Data shown are representative of 4 different experiments that produced similar results. (G) Multilineage contribution of donor cells in recipients at 13 weeks after transplantation (n = 9). (H) Ikaros-overexpressing HSCs had diminished repopulation capacity in secondary transplantation. Fifteen hundred CD45.2 GFP⁺Lin⁻Sca-1⁺Kit⁺ cells isolated from primary transplanted mice as described in panel F were cotransplanted with 10⁵ CD45.1 bone marrow competitors into lethally irradiated CD45.1 recipients for secondary competitive repopulation analysis. Donor engraftment at 3, 8, and 16 weeks after transplantation is shown (*P < .05, n = 5).