Figure 5.

Cytosolic ssDNA primes cDKO HSPC exit from quiescence, and p53 activation leads to apoptotic cell death in cDKO BM. (A) Immunofluorescent staining shows ssDNA (green) presence in the cytoplasm of LKS⁺ cells (left panel) and immunohistochemistry staining of cytosolic ssDNA (brown) on BM sections of cDKO mice on day 3 after 4 mg of TAM. Images were acquired at ×100 magnification. (B) Cell cycle analysis of BM LKE⁺ cells in a time course of 4 days after cDKO. (C) Histological analysis of H&E and immunohistochemistry staining of Ki67, p53, and ApopTag. Images were acquired at ×40 magnification. (D) Western blot showing Ssb1, Ssb2, and p53 levels in cDKO BM samples on D0 and D4 after 4 mg of TAM. (E) LKS⁺ cells (CD45.2) were isolated from donors, transduced with shLuc-GFP or shP53-GFP, mixed in a 1:1 ratio with competitor BM (CD45.1), and transplanted into irradiated recipient mice. cDKO was induced in 4 weeks posttransplantation. Peripheral blood was monitored to measure bone marrow chimera percentage in recipient mice every 4 weeks post-TAM (cDKO) or vehicle control administration until week 16. (F) Hoxb8-immortalized LKS⁺ cells were transduced with control (shLuc-GFP) or p53 (shP53-GFP) shRNAs. Apoptosis was measured 5 days after in vitro 4OHT induction of cDKO. Statistical analysis represents 2-way ANOVA. Each point represents an individual mouse/biological replicate. See also supplemental Figure 5. *P < .05; **P < .01; ***P < .001. DAPI, 4′,6-diamidino-2-phenylindole; IHC, immunohistochemistry; NS, not significant; Veh, vehicle.