Deletion of p65 greatly reduces the repopulation ability of HSCs. (A) 1 × 10⁶ test cells (CD45.2) from wild-type or p65^hem−/− mice were injected into lethally irradiated recipients along with 1 × 10⁶ healthy competitor cells (CD45.1). Engraftment was determined by virtue of CD45.1 and CD45.2 markers on peripheral blood cells (and bone marrow, data not shown) of recipients 20 weeks after transplant. (B) Varying numbers of test cells (CD45.2) from wild-type or p65^hem−/− mice were injected into lethally irradiated recipients along with 2 × 10⁵ healthy competitor cells (CD45.1). Engraftment was determined by virtue of CD45.1 and CD45.2 markers on peripheral blood cells (and bone marrow, data not shown) of recipients 20 weeks after transplant. (C) The frequency of functional HSCs was calculated using L-Calc software. (D) 2 × 10⁷ whole bone marrow cells from wild-type or p65^hem−/− mice were injected into the lateral tail vein of lethally irradiated mice. Engraftment of donor cells in the bone marrow of recipients was analyzed 48 hours after transplant (n = 4). (E) Apoptosis of HSPCs from p65^hem−/− mice or littermates was analyzed by Annexin V staining (n ≥ 7). (F) Wild-type or p65^hem−/− mice were injected with EdU 18 hours prior to harvest, and the proliferative index of HSPCs was analyzed by flow cytometry (n = 5). (G) Bone marrow was isolated from primary recipients reconstituted with 10⁶ test cells and 10⁵ competitor cells (B), and then 3 × 10⁶ cells were injected into lethally irradiated secondary recipients. Engraftment was monitored 20 weeks after transplant. (n = 2 donors per group with 85% to 90% CD45.2⁺ cells, 10 recipients per group). *P ≤ .005.