DN-JAK2 but not DN-STAT5 inhibits EPO-mediated survival.

(A) DP16.1/p53ts cells were transfected with expression plasmids containing DN-JAK2: JAK2-DK encodes JAK2 with mutations in the kinase domain and JAK2Δ829 encodes JAK2 with a truncated kinase domain; pEF-BOS is the empty vector. After 12 hours at 32°C in the presence or absence of EPO, cultures were fixed and the proportion of cells undergoing apoptosis was measured by the TUNEL assay. The data presented are representative of 2 independent experiments. NT indicates cells that were not transfected. (B) DP16.1/p53ts cells were cotransfected with vectors expressing CD20 together with DN-STAT5 or empty vector (pcDNA3). Cells were cultured as described in the legend to Panel A, fixed, and stained for CD20 expression and for apoptosis with PE-conjugated annexin V, and analyzed by flow cytometry. The percentage of CD20⁺ cells undergoing apoptosis is presented as the means ± SEM (n = 3). (C) Untransfected DP16.1 cells (left panel), DP16.1 cells transfected with CD20 and empty pcDNA3 vector (middle panel), or DP16.1 cells transfected with CD20 and DN STAT5 (right panel) were treated with EPO for 15 minutes, fixed, and stained with phospho-specific antibodies against STAT5 (Tyr694). Cells expressing phosphorylated STAT5 were detected by flow cytometry using an anti–rabbit IgG conjugated to FITC. The histograms show the relative fluorescence for FITC of cells that were either left untreated (filled histogram) or treated with EPO (empty histogram) 24 hours after transfection.