Jmjd6-deficient HSPCs display molecular signatures of functional HSC decline, which can be rescued by removing ROS. (A) GSEA showing upregulated and downregulated pathways in LSKs from 8- to 12-week-old Jmjd6^cKO mice compared with LSKs from 8- to 12-week-old Jmjd6^CTL mice (n = 4). (B) GSEA plots for stabilization of p53, apoptosis via p21/p53 axis, and OXPHOS based on analysis of gene expression changes, using Jmjd6^cKO LSK cells for upregulated pathways (n = 4). (C) OCR in Jmjd6^cKO and Jmjd6^CTL BM c-Kit⁺ cells under basal conditions and maximal OCR. Maximal OCR was achieved by the sequential addition of oligomycin (ATPase inhibitor), FCCP (mitochondrial uncoupler), and rotenone and antimycin A (complex I and III inhibitors, respectively) (n = 3). (D) ROS levels in c-Kit⁺Jmjd6^cKO and Jmjd6^CTL BM cells after a 1-hour incubation with CellROX reagent. Data represent mean fluorescence intensity (MFI) (n = 8-9). (E) Experimental design. BM cells from 8- to 10-week-old Jmjd6^cKO and Jmjd6^CTL mice were placed in CFC assays with 5 mM NAC or PBS control. CFC1 colonies were counted and scored 10 days after plating and replated into CFC2. CFC2 colonies were counted after 10 days in culture. (F) CFC1. G, granulocyte; GEMM, granulocyte, erythroid, macrophage, or megakaryocyte; GM, granulocyte and monocyte/macrophage; M, monocyte/macrophage; (n = 4). (G) CFC 2 (n = 4). (H) Experimental design. Eight- to 12-week-old Jmjd6^cKO and Jmjd6^CTL mice were administered a daily dose of NAC (100 mg/kg) via intraperitoneal injection, as well as in drinking water (1 mg/mL). Hematopoietic compartments were analyzed following 10 days of treatment. Total cell numbers of HSCs (I) and CLPs (J) in BM from Jmjd6^cKO and Jmjd6^CTL mice after 10 days of NAC treatment (n = 4-5). Data are mean ± SEM. *P < .05, **P < .01. NS, not significant.