Healthy bone marrow donors have lower levels of total STAT5 expression and STAT5 phosphorylation than MPD samples. Cells from bone marrow CD34+ cells from a subject with ET (769, V617F+) and a healthy donor (NBM6) were grown in parallel for 9 days in the presence of IL-3 (10 ng/mL), IL-6 (10 ng/mL), SCF (25 ng/mL) and EPO (2 U/mL starting on day 5). (A) Cells were counted and replated at 5 × 105 cell/mL and then exposed for 24 hours to lestaurtinib at different concentrations, lysed, and expression of phosphorylated STAT5 and total STAT5 was assayed by Western blotting. (B) After expansion, cells were washed twice in PBS and replated in media lacking all cytokines for 4 hours and then restimulated for 10 minutes with EPO (100 U/mL), TPO (100 ng/mL), or no cytokine control. Cells were collected and lysed. Lysates were analyzed for phospho-STAT5 and total STAT5 expression.
