Loss of Ott1 does not cause increases in HSC apoptosis, proliferation, or senescence. (A) Flow cytometric measurement of apoptotic (annexin V⁺/7-AAD⁻) and dead/apoptotic (annexin V⁺/7-AAD⁺) percentage in LT-HSCs (Lin⁻Sca-1⁺c-Kit⁺CD34⁻) and ST-HSCs (Lin⁻Sca-1⁺c-Kit⁺ CD34⁺) from BM harvested from Ott1^flox/null Mx1-cre (KO) or littermates possessing a WT Ott1 allele (control) 6 weeks after pIpC injection. (B) Cell-cycle analysis of LT-HSCs (Lin⁻Sca-1⁺c-Kit⁺CD34⁻). KO and control BM obtained as in panel A were stained with Hoechst 33342/pyronin Y, and flow cytometry was performed to quantify G₀, G₁, and S-G₂-M phases. Representative flow panels above of control (left) and KO (right). (C) Cell-cycle analysis as in panel B for ST-HSCs (Lin⁻Sca-1⁺c-Kit⁺CD34⁺; n = 5, control; n = 4, KO). (D) SA–β-Gal expression in sorted control and Ott1 KO LSK cells. Cytospins of sorted cells were stained for SA–β-Gal and examined under light microscopy. A total of 100 cells were counted from each group, and the experiment was performed in duplicate. No β-Gal–positive cells were observed. Black bars represent 10 μm. (E) Quantitative real-time PCR from total RNA of sorted control and Ott1 KO LSK cells. Bars represent mean of Ott1 KO relative to control (n = 3).Graphs represent mean values, and error bars represent SD.