Ott1-deleted HSCs have a gene expression profile similar to aged HSCs with associated physiologic changes. (A-C) GSEA comparing genes up-regulated in sorted LSK cells from Ott1 KO mice compared with WT controls with published gene sets up-regulated in aged HSCs. (A) GSEA using Chambers et al¹⁷  1667 genes up-regulated with age in HSCs. (B) GSEA using a 79-gene subset of (A) restricted to genes expressed in HSCs versus whole BM.¹⁷  (C) GSEA comparing Ott1 KO LSK with Rossi et al aged HSC profile.³⁵  (D) Localization of p65 NF-κB subunit. Cytospins of sorted control and Ott1 KO LSK cells were stained with rabbit anti-p65 and anti–rabbit-AF488 secondary antibody with DAPI nuclear stain. Cells were imaged by confocal microscopy. A minimum of 30 cells were assessed from each group, and the experiment was performed in duplicate. (E) MFI of DCF-DA–labeled cells from Ott1 KO and control BM. LT-HSCs (Lin⁻Sca-1⁺c-Kit⁺CD34⁻), ST-HSCs (Lin⁻Sca-1⁺c-Kit⁺CD34⁺), and progenitors (Lin⁻Sca-1⁻c-Kit⁺). Representative histograms of flow above. (F) Measurement of phospho–γ-H2AX levels in Ott1 and control LT-HSC, ST-HSC, and progenitor populations. Cells stained for intracellular phospho–γ-H2AX using phospho-specific antibody. Representative flow histograms above. Graph bars represent mean of MFI, and error bars represent SD. Control, n = 4; Ott1 KO, n = 3. (G) Phospho-p38Mapk staining of Ott1 KO and control LSK cells. KO or control BM as in panel A was either untreated (baseline) or grown 48 hours in IL-3, IL-6, and SCF (cytokine). Cytospins of sorted LSK cells were stained with anti–phospho-p38Mapk and AF488 secondary antibody and then imaged by confocal microscopy with DAPI nuclear counterstain. Minimum of 40 cells scored. White bar represents 10 μm.