c-Myc regulates multiple pathways related to HSC functions. (A) A volcano plot showing fold changes for differentially expressed genes in LT-HSCs from WT (c-Mycfl/+) and c-Myc HET (c-Mycfl/+Mx1Cre) mice. The experiments were performed in triplicate. (B) Bar graph showing gene ontology biological processes significantly affected in c-Myc HET HSCs. (C) Enrichment plots of selected gene sets from GSEA. Expression data with 20 000 transcripts was used for analysis. (D) qPCR analysis of the expression of selected genes in LT-HSCs. Gene expression was initially normalized to actb expression. Values represent the fold changes in gene expression relative to that in control HSCs. (E) The c-Myc potential binding sites on the promoter region(s) of Jmjd3, Nr4a1, and Nr4a2. (F) Luciferase reporter assays. 293T cells were transfected with Jmjd3, Nr4a1, or Nr4a2 promoter luciferase constructs and control vector PIG or c-Myc–expressing vector. (G) ChIP assay of the binding of endogenous c-Myc to the promoter region(s) of Jmjd3, Nr4a1, and Nr4a2 in Lin− BM cells; IgG served as the negative control. (H-J) Total HSC number (H), the frequency of apoptotic HSCs (I) and the frequency of quiescent HSCs (J), as determined by flow cytometric analysis, in mouse recipients of transplanted WT BM cells expressing MSCV vector, or c-Myc HET BM cells expressing MSCV vector, JMJD3, Nr4a or Nr4a2 (n = 3-6). *P < .05; **P < .01; ***P < .001, by Student t test.