Comparative analysis of senescence-associated genes and a limited role of Ews in stromal cells' capacity to support hematopoiesis. (A) Top panel: total RNA was prepared from freshly isolated Ews^+/+ and Ews^−/− Lin⁻ BM cells and then subjected to quantitative PCR analysis. Bottom panel: 5 × 10⁵ BM cells from Ews^−/− mice and WT littermates were transplanted into irradiated recipients. Two months after transplantation, LSK cells derived from WT or KO donors in the recipients were sorted and subjected to quantitative PCR analysis. The expression level in WT cells was arbitrarily set to 1. The fold change in expression of each gene was calculated using the ΔΔCt method. An asterisk indicates a statistically significant difference (2-tailed Student t test; *P = .03; **P = .006). (B) Left panel: LSK cells were sorted from Ews^−/− and WT littermates and then subjected to quantitative PCR analysis. The expression level in WT cells was arbitrarily set to 1. Right panel: immunoblot analysis of endogenous Ezh2 protein expression in E14.5 fetal liver cell extracts from Ews^−/− and WT littermates. (C) Primary BM stromal cells were prepared from Ews^+/+ (WT), Ews^+/− (HT) and Ews^−/− (KO) littermates. Normal BM cells were seeded onto the stroma cells. The CAFC assay was performed as described in “CFC and CAFC assays.”