Asrij depletion significantly reduces quiescence and increases proliferation of HSCs. Floxed and KO cells were analyzed in all cases and are indicated in red and light green, respectively. (A) Representative Hoescht 33342/Pyronin-Y flow cytometric plots and graphs showing the cell-cycle distribution of HSPCs, LT-HSCs, ST-HSCs, and MPPs (n = 3 mice per genotype). (B) Cell proliferation analysis using Ki-67/7-AAD staining of HSPCs, LT-HSCs, ST-HSCs, and MPPs (n = 3 mice per genotype). (C) Representative images of OP9-HSPC cocultures showing colonies (red arrows) formed by HSPC (LSK-CD34⁻ and LSK-CD34⁺) subpopulations. Bar represents 50 µm. Graphs quantify number of colonies formed and number of cells/colony (n = 3 mice per genotype). (D) Flow cytometric analysis and quantification of the HSPC frequencies in spleen and PB (n = 4 mice per genotype). CFU-C assay to test the differentiation potential of (E) BM cells and (F) splenocytes harvested from 6-mo-old mice under erythromyeloid promoting conditions (n = 3 mice per genotype). (E) Graphs show relative distribution of total, BFU-E, CFU-GM, and CFU-granulocyte/erythroid/macrophage/megakaryocyte colonies scored based on their morphology and colony diameter. (F) Graphs show colony percentage and colony diameter of CFU-GMs formed by splenocytes. Statistically significant differences identified using ANOVA: single factor analysis are indicated. Error bars denote standard error of mean. *P < .05 and **P < .01.