Ott1-deleted HSCs cannot be rescued with antioxidant treatment and have increased mitochondrial mass. (A) Schema for determining whether NAC is capable of rescuing the maintenance defect in Ott1-deleted HSCs. Unexcised CD45.2 Ott1^flox/null Mx1cre and control BM were transplanted into lethally irradiated CD45.1 recipients in a 4:1 ratio with CD45.1/CD45.2 heterozygous WT competitor BM. Four weeks were allowed for stable engraftment; then the 2 cohorts of mice were each split into 2 arms and received either 200 mg/kg NAC or saline placebo intraperitoneally daily for the remainder of the experiment. After 3 days of NAC treatment, the mice were bled to assess baseline engraftment of donor marrow through CD45.1/CD45.2 ratios in the Mac1⁺/Gr1⁺ peripheral blood population and then treated with pIpC. At 3 weeks after pIpC, peripheral blood and BM were obtained to analyze chimerism. (B) Precentage of donor chimerism of peripheral blood neutrophils from mice transplanted with BM Ott1^flox/null Mx1-cre (KO) or WT Ott1 (control) plus competitor pre-pIpC and after pIpC/NAC for 3 weeks. (C) Peripheral blood neutrophil donor chimerism at 3 weeks after pIpC of Ott1 KO and control donor treated with NAC or saline. (D) Precentage of donor chimerism of LT-HSC (Lin⁻Sca-1⁺c-Kit⁺CD34⁻) populations from Ott1 KO or control 3 weeks after pIpC and NAC or saline treatment. Donor identified by detection of CD45.2/CD45.2 markers by flow cytometry (n = 4). (E) MFI of Mitotracker deep red of Ott1^flox/null Mx1-cre (Ott1 KO) or littermates possessing a WT Ott1 allele (control) 6 weeks after pIpC of LT-HSC (Lin⁻Sca-1⁺c-Kit⁺CD34⁻), ST-HSC (Lin⁻Sca-1⁺c-Kit⁺CD34⁺), and progenitor (Lin⁻Sca-1⁻c-Kit⁺) populations. Representative flow histograms at right. Control (gray line), n = 4; Ott1 KO (black line), n = 3. Graph bars represent mean values, and error bars represent SD.