Mortalin is abundantly localized in the mitochondria of HSPCs. (A) qRT-PCR analysis of mortalin in CD34⁻ LSK, CD34⁺ LSK, and Lin⁻ cell populations. β-actin was used as an endogenous control. Data represent the mean ± standard deviation(SD) (n = 4; ***P < .001). (B) Western blot analysis of mortalin and β-actin in total cell extracts derived from freshly isolated LSK, Lin⁻, and MNC populations. (C) Fluorescence microscopy images of freshly isolated CD34⁻ LSK, LSK, Lin⁻, and Lin⁺ cells stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue) and an anti-mortalin Ab (green). The smaller boxes show higher power views of the areas denoted by arrowheads. Scale bar, 20 μm. (D) Western blot analysis of mortalin in cytoplasmic, mitochondrial, and nuclear fractions prepared from Lin⁻ BM cells. Hsp90, Hsp60, and Histone H3 were used as cytoplasmic, mitochondrial, and nuclear markers, respectively, to monitor the purity of the subcellular fractions. (E) Fluorescence microscopy images of freshly isolated CD150⁺ LSK and LSK cells stained with an anti-mortalin Ab (red), Mito Tracker (a cell-permeable membrane marker, green), and DAPI (blue). Scale bar, 5 μm. (F) Electron microscopy images of ultrathin sections of Lin⁻ BM cells. N, nucleus; C, cytoplasm; Mt, mitochondria. Arrows indicate immunogold-labeled mortalin. Magnified images show details in single mitochondria. Scale bar, 200 nm.