Lestaurtinib inhibits growth and JAK/STAT signaling in cultured erythroid precursors. Effects of lestaurtinib on proliferation (A, C, E, G) and STAT5 phosphorylation (B, D, F, H) in erythroid precursors derived from CD34+ progenitors purified from peripheral blood of MPD patients (A-F) and from a bone marrow of the healthy subject (G, H) were evaluated. Cultures were counted and replated at 5 × 105 cells/mL and then treated with increasing concentrations of lestaurtinib, as indicated, for 48 to 72 hours and cell proliferation was analyzed by XTT assay (A, C, E, G). To assess effects of lestaurtinib on JAK/STAT signaling (B, D, F, H), cultures were treated with increasing concentrations of lestaurtinib for 1 hour, and activation of STAT5 was analyzed by Western blot using phospho-specific STAT5 antibody. Blots were stripped and reprobed with a total STAT5 antibody. The status of the V617F mutation was determined in mononuclear cells by allele-specific PCR and BsaX1 restriction analysis. Stripping and reprobing of blot in H confirmed equal protein loading (data not shown). Vertical lines have been inserted to indicate a repositioned gel lane. Error bars in panels A, C, E, and G represent SE.
