Deletion of Setdb1 leads to rapid depletion of HSPCs in BM. (A) Deletion of Setdb1 in Cre-ERT;Setdb1^fl/fl BM Lin^-c-Kit⁺ hematopoietic progenitor cells were confirmed by genomic PCR 2 weeks after the first injection of tamoxifen. “WT,” “floxed,” and “∆” alleles indicate the wild-type Setdb1 allele, floxed Setdb1 allele, and floxed Setdb1 allele after the removal of exons 15 and 16 by Cre recombinase, respectively. (B) RT-PCR of Setdb1 expression in LSK HSPCs 2 weeks after the first injection of tamoxifen. mRNA levels were normalized to Hprt1 expression, and relative expression levels are shown as the mean ± SD of triplicate analyses. (C) Survival curve of recipient mice repopulated with BM cells from Cre-ERT (WT) and Cre-ERT;Setdb1^fl/fl (Setdb1^Δ/Δ) mice after the initial injection of tamoxifen (n = 10). BM cells from 8-week-old Cre-ERT and Cre-ERT;Setdb1^fl/fl mice were transplanted into lethally irradiated recipient mice without competitor cells. At 4 weeks after transplantation, recipient mice were injected with tamoxifen for 5 consecutive days. (D) Representative hematoxylin and eosin staining of BM sections from WT and moribund Setdb1^Δ/Δ mice in (C). (E) The proportions of Mac-1⁺ myeloid, B220⁺ B, and CD4⁺ or CD8⁺ T cells among CD45.2⁺ donor-derived hematopoietic cells in PB from WT and Setdb1^Δ/Δ mice (n = 4) 2 weeks after the first injection of tamoxifen. Data are shown as the mean ± SD. (F) Absolute numbers of total BM cells, CD34⁻LSK HSCs, CD34⁺LSK multipotent progenitor cells, common myeloid progenitors, GMPs, and megakaryocyte/erythrocyte progenitors in the unilateral femur and tibia of WT and Setdb1^Δ/Δ mice (n = 4) 2 weeks after the first injection of tamoxifen. Data are shown as the mean ± SD. (G) Percentage of Annexin V⁺ cells in BM LSK and LK cells in WT (n = 4) and Setdb1^Δ/Δ mice (n = 6) 9 days after the first injection of tamoxifen. Data are shown as the mean ± SD. (H) Representative flow cytometry profiles of the incorporation of Pyronin Y by BM CD45.2⁺ LSK cells 2 weeks after the first injection of tamoxifen (left). The percentage of Pyronin Y⁺ cells in LSK cells in WT and Setdb1^Δ/Δ mice (n = 4) are shown as the mean ± SD (right). (I) Cell cycle status of LSK and Lin⁻c-Kit⁺ Sca-1⁻ (LKS⁻) committed progenitor cells evaluated by BrdU short-labeling assays. BrdU was administered 12 and 24 hours before the analysis to mark cells that entered S phase. Data are shown as the mean ± SD (n = 6). (J) Colony-forming assays with LSK cells from WT and Setdb1^Δ/Δ mice 2 weeks after the initial injection of tamoxifen. The absolute numbers of colonies with the indicated size per 200 LSK cells are shown as the mean ± SD of triplicate cultures. (K) Competitive reconstitution assays. BM cells from 8-week-old CreERT and CreERT;Setdb1^fl/fl mice (2 × 10⁶ cells) were transplanted into lethally irradiated recipient mice with competitor BM cells (1 × 10⁶ cells). At 8 weeks after transplantation, recipient mice were injected with tamoxifen for 5 consecutive days. The chimerism of CD45.2⁺ donor-derived cells in all CD45⁺ cells and in myeloid, B, and T cells in the PB of recipient mice is shown as the mean ± SD (n = 5). *P < .05; **P < .01; ***P < .001; ns, not significant.