Aged HSCs exhibit decreased expression of Lin28b-Hmga2-Igf2bp2 mRNAs and reduced activity of the PI3K/AKT/mTOR pathway. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR), immunofluorescence, and fluorescence-activated cell sorting (FACS) analyses of freshly isolated total CD150⁺ (high and low) HSCs (CD150⁺CD34⁻LSK) and MPPs (CD34⁺LSK) from young mice (range: 3-6 months) and old mice (range: 22-28 months). (A-B) The relative mRNA expression of Lin28b, Hmga2, and Igf2bp2 (relative to Actb) was analyzed by qRT-PCR in HSCs (A) and MPPs (B). Five to 14 mice per group were analyzed in 2 independent experiments. For each gene and cell type, one sample of young wild-type mice was set to 1 and used as callibrator. Data were log₂ transformed and analyzed by Welch’s t test. (C-F) Representative micrographs and quantification of the MFI of p-AKT staining in HSCs (C-D) and MPPs (E-F). The MFI of young mice was normalized to 1 for each of the 2 cell populations. A total of 10 to 13 mice per age group were analyzed in 2 independent experiments. Statistical significance was assessed by Welch’s t test. (C,E) Bars represent 10 μm. (G-H) Quantification and representative FACS profiles of the fluorescence intensity of p-mTOR in HSCs and MPPs. (G) The mean of the MFI of HSCs from young mice was set to 1. A total of 12 to 14 mice per group were analyzed in 2 independent experiments. Statistical significance was assessed by 2-way analysis of variance on log-transformed data followed by pairwise t tests with Sidak’s correction for multiple comparisons. (H) Representative FACS profiles of HSCs and MPPs from young and old mice. (A-B,D,F-G) Horizontal lines represent the mean of the indicated group. MFI, mean fluorescence intensity; ns, nonsignificant.