FIGURE 5.

Expression of Mnk1 and Mnk2 during normal human bone marrow erythroid differentiation and their roles as mediators of Type I IFN-dependent suppression of JAK2V617F transformed CD34⁺-derived human erythroid progenitors. A and B, bone marrow-derived human CD34⁺ cells were allowed to differentiate toward the erythropoietic lineage, using the methodology described under “Materials and Methods.” mRNA expression of Mnk1 (A) and Mnk2 (B) genes was assessed at the indicated days of hematopoietic cell differentiations by quantitative RT-PCR (Taqman), using GAPDH for normalization. Data are expressed as fold induction over corresponding samples obtained at day 3 and represent means ± S.E. of three independent experiments. C, expanded CFU-E cells were nucleofected with JAK2wt-IRES-GFP or JAK2V617F-IRES-GFP expressing vectors for 48 h. Equal amounts of protein were separated by SDS-PAGE and then immunoblotted with antibodies against JAK2 or GAPDH. D, normal CD34⁺ bone marrow-derived cells were transfected with JAK2wt-IRES-GFP or JAK2V617F-IRES-GFP vectors, respectively. These cells were then incubated in the presence or absence of IFNα, along with DMSO or Mnk-I, in clonogenic assays in methylcellulose as indicated. Myeloid (CFU-GM) and early erythroid (BFU-E) progenitor colonies were scored after 14 days in culture. Data are expressed as percentage control (CTRL) colony formation from untreated cells and represent means ± S.E. of four independent experiments.