ATMIN-deficient mice have a reduced CMP compartment due to increased apoptosis. Black bars/symbols represent Atmin^f/f, and open symbols/gray bars represent Vav-Atmin^∆/∆, unless otherwise indicated. (A) Dichlorodihydrofluorescein diacetate (DCFDA) stains for ROS detection showing a mild increase in the percentage of ROS-positive cells in ATMIN-deficient CMPs (n = 3/genotype). (B) Treating total BM cells with the antioxidants N-acetylcysteine (NAC) or γ-glutamylcysteinylethyl ester (not shown) was unable to rescue the myeloid colony formation defect of Vav-Atmin^∆/∆ cells (n = 5/genotype). (C) Expression of key antioxidant genes was unchanged between ATMIN-deficient and control CMPs. Expression values were determined by qRT-PCR and were relative to the mean (n = 3-5/genotype). (D-F) In ATMIN-deficient CMPs compared with control CMPs, no significant increase in pKap1 levels (indicative of ATM activation) (D), upregulation of genes required for DNA oxidation repair (E), or changes in the indicated CMP developmental factors (F) were detected. pKap1 levels were determined by intracellular flow cytometry analysis (mean fluorescence intensity [MFI] ratios are shown), and qRT-PCR was used to determine the expression of the indicated genes (n = 5-6/genotype). The frequency of late apoptosis was significantly increased (G) and Dynll1 expression was drastically reduced (H) in CMPs lacking ATMIN compared with control cells (n = 4-6/genotype). (I) Representative cell images captured by an ImageStream flow cytometer in green and red channels, followed by their respective composite images, showing higher colocalization of mitochondria (red) with Bim (green) staining in Vav-Atmin^∆/∆ CMPs. (J) Representative MitoTrackerRed-CMXRox and Bim similarity staining score histograms for Atmin^f/f-derived (black) and Vav-Atmin^∆/∆-derived (open) CMPs respectively. A score above value 2 (R1) indicates translocation of Bim into mitochondria. (K) Percentage of Bim translocation in CMPs (n = 3/genotype). The different BM hematopoietic stem and progenitor populations were defined as follows: LT-HSC, Lineage⁻Sca-1⁺cKit⁺ (L⁻S⁺K⁺)CD34^−/loFlt3⁻; short-term HSC, L⁻S⁺K⁺CD34⁺Flt3⁻; multipotent progenitor, L⁻S⁺K⁺CD34⁺Flt3⁺; CMP, L⁻S⁻K⁺IL7R⁻CD34⁺FcγR⁻; granulocyte (G) monocyte (M) progenitor, L⁻S⁻K⁺IL7R⁻CD34⁺FcγR⁺; megakaryocyte-erythroid progenitor, L⁻S⁻K⁺IL7R⁻CD34⁻FcγR⁻; and common lymphoid progenitor, L⁻S⁻K^loIL7R⁺. Mean and standard deviation values are shown. *P < .03; ***P < .0003. DAPI, 4′,6-diamidino-2-phenylindole; HSC, hematopoietic stem cell; LT, long-term; mRNA, messenger RNA; qRT, quantitative reverse transcription.