M40^−/− HSCs show decelerated cell cycle progression, increased quiescence, and downregulation of gene expression associated with cell division. (A) BrdU incorporation analysis in SLAM LSK cells. WT and M40^−/− mice were either fed with water containing BrdU for 7 days or intraperitoneally injected 2 hours prior to sacrifice. Representative flow cytometric plots of SLAM HSCs and histograms depicting BrdU labeling are shown. BrdU⁺ fractions were quantified (mean ± SD); n = 6. (B) Sorted SLAM LSK HSCs were stained with Py and Ho, and representative flow cytometric plots are shown. The quiescence G0 populations defined as Py⁻Ho⁻ are indicated (mean ± SE); n = 4. P values were calculated by the Student t test. *P < .01; **P < .05. (C) Enrichment plot of GSEA analysis using WT vs M40^−/− HSC expression data against a cell cycle signature from the MSigDB c2.cp database. (D) RT-qPCR analysis of cell cycle regulatory genes in WT and M40^−/− HSCs. Relative expressions to GAPDH (mean ± SE) are shown. P values were calculated by the Student t test. *P < .001; **P < .02. FDR, false discovery rate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ho, Hoechst; NES, normalized enrichment score; Nom, nominal; Py, Pyronin Y; SD, standard deviation; SE, standard error.