Decreased expression of niche factor, Cxcr4, and impaired homing may be responsible for Runx1^−/− stem cell exhaustion. (A) Left panel: flow cytometric analysis of Cxcr4 expression on KSL cells from Runx1^+/+ (n = 4) and Runx1^−/− (n = 3) mice. Right panel: qRT-PCR analysis of expression of Cxcr4 in KSL fraction of Runx1^+/+ and Runx1^−/− BM cells. Statistical difference using unpaired Student t test is given at the bottom. (B) Expression of Cxcr4 in c-Kit⁺GFP⁺ cells from wild-type BM cells transfected with mock MIG vector (dashed line) or RUNX1-ETO (solid line). One representative result of 2 experiments is shown. (C) Structure of the CXCR4 promoter luciferase reporter construct. The 2 arrowheads represent the positions of 2 consensus Runx1 binding sites on the human CXCR4 promoter. Graph represents the result of luciferase assay, showing transcriptional activity of wild-type RUNX1 (WT) or its mutant form R174Q (MT) with (+) or without CBFB, on CXCR4 promoter. (D) Graph showing percentage of CFSE-stained Runx1^+/+ or Runx1^−/− BM cells found in the recipient BM (n = 4 and 6, respectively), 16 hours after transplantation. Statistical difference using unpaired Student t test is given at the bottom. (E) Graphic representation of colony assay of 20 μL of PB from Runx1^+/+ (n = 4) and Runx1^−/− (n = 4) mice. (F) FACS analysis of spleen KSL fraction in Runx1^+/+ and Runx1^−/− mice. One representative flow cytometry profile from 2 experiments is shown. (G) Graph showing absolute number of BrdU⁺ KSL cells per 1 million BM cells analyzed from Runx1^−/− (n = 3) and Runx1^+/+ mice (n = 3). Statistical difference using unpaired Student t test is given at the bottom.