C/EBPβ isoforms play different roles in the regulation of HSPCs. (A) EML cells were transduced with control (pGCDNsam-IRES-Kusabira-Orange [KuO]) or LIP, LAP, or LAP* expression vectors (pGCDNsam-LIP-KuO, pGCDNsam-LAP-KuO, or pGCDNsam-LAP*-KuO), and KuO⁺ cells were subjected to analysis. Cell numbers are expressed as fold change relative to the number on day 3 (n = 4 per group and per time point, representative of 2 independent experiments). (B) BrdU incorporation by EML cells 3 days after transduction of control, LIP, LAP, or LAP* expression vector (n = 5 per group). In each experiment, the frequency of BrdU⁺ cells in the control vector-transduced cells was set to 1, and the values from 5 independent experiments were statistically analyzed using the 2-tailed paired Student t test. (C) Representative flow cytometric analysis of EML cells 5 days after transduction of the control, LIP, LAP, or LAP* expression vector. (D) Schematic illustration of the BM transplantation experiments in combination with retroviral transduction. (E) Frequencies of BM c-kit⁺Sca-1⁺lineage⁻ cells (KSLs) among control vector- and LIP-transduced WT BM cells 3 weeks (i) or 16 weeks (ii) after transplantation (n = 5 per group, representative of 2 independent experiments). LT-HSCs were defined as CD135⁻CD150⁺CD48⁻ KSL cells. (F) Cell-cycle statuses of control vector- and LIP-transduced WT LT-HSCs (CD150⁺CD48⁻ KSL cells) at 3 or 16 weeks after transplantation. Frequencies of cells in G0 phase are shown (n = 4 per group, representative of 2 independent experiments). (G) Frequencies of indicated HSPCs subsets among control vector- and LIP-transduced WT BM cells 3 weeks (i) or 16 weeks (ii) post-transplantation (n = 5 per group, representative of 2 independent experiments). LT-HSCs were defined as CD135⁻CD150⁺CD48⁻ KSL cells. (H) PB CD11b⁺ cells subsets among control vector- and LIP-transduced WT BM cells 3 weeks (i) or 16 weeks (ii) after transplantation or 2 weeks after 5-FU treatment at 16 weeks after transplantation (iii) (n = 6-10 per group, representative of 2 independent experiments). Data are presented as means ± SD. *P < .05; **P < .01; and ***P < .001 (determined by the 2-tailed Student t test).