Figure 4: The ROSlow subset bears a chemoresistant phenotype and can establish leukemia in NOD/SCID mice. (a) Primary engraftment of sorted ROSlow and total CD34+ AML cells from primary patient samples (n=3) in the bone marrow of NOD/SCID mice (left) Each animal is shown as a dot and animals from the same sample are shown as similar dots. Representative flow cytometry plots of the detection of human CD45/CD33 double positive cells in the bone marrow of a NOD/ SCID mice transplanted with either ROSlow or total AML blasts and harvested 12 weeks after transplantation (right). Horizontal bars indicate mean. (b) Cumulative results and representative plots (c) of cell cycle analysis in flow-sorted total and ROSlow CD34+ cells (n=5). A significantly higher fraction of ROSlow cells resides in G0 phase, as compared to bulk leukemia cells. By contrast, the latter are more frequently found in G1 and S/G2/M phases. (d) ROSlow cells exhibit increased in vitro chemoresistance in both daunorubicin (DNR, 0.5–2 μΜ, n=4) and cytarabine (AraC, 2.5-10 μΜ, n=4). Viability was normalized to the percentage of viable cells after 24 hours in the control (no drug) cultures and is presented as mean ± SEM of triplicates. (e) Basal and potentiated phospho-STAT5 expression on sorted ROSlow and total AML blasts (n=4). GM-CSF-induced phosphorylation of STAT5 was significantly higher in ROSlow cells, whereas no differences were observed after G-CSF stimulation. Data are expressed as mean ± SEM; *P<0.05 by paired Student’s t-test