Ews deletion drivescell-cycleprogression in hematopoieticstemandprogenitor cellpopulations and modulates progenitor cell activity. (A) Flow cytometric analysis of PY (Pyronin-Y) and HO (Hoechst 33342) staining of Lin⁻Sca1⁺c-Kit⁺ and Lin⁻Sca1⁻c-Kit⁺ cells from Ews KO and WT littermate controls. The percentage of cells in each phase of the cell cycle is summarized in the accompanying table. Data shown are representative of 2 independent experiments. (B) CFU-S assay. At day 12, spleens were removed and fixed in Bouin fixative and CFU-S colonies were counted. The CFU-S activity of Ews^−/− (KO) and Ews^+/+ littermate control (WT) BM cells was assessed before transplantation and after 2 rounds of transplantation (after transplantation). Of note, when Ews^−/− BM cells were subjected to transplantation, their CFU-S activity was markedly decreased and showed a lower CFU-S activity compared with that of WT. Data are presented as mean values ± SD. (C) CAFC assay. The CAFC activity of Ews^−/− (KO; white bar) and Ews^+/+ littermate control (WT; black bar) BM cells was assessed before transplantation and after 2 rounds of transplantation (after transplantation). Of note, Ews^−/− BM cells did not have measurable week-4 CAFC activity after BM transplantation, which is indicative of progenitor cell exhaustion. A total of 48 replicate wells were prepared per dilution and evaluated. Any well that had a colony of more than 6 cobblestone cells growing underneath the stromal cell layer was scored as positive. The y-axis denotes the percentage (%) of positive wells. Data are presented as mean ± SD. *Statistically significant difference (2-tailed Student t test, n > 4).