Functional analysis of MPL mutants. (A) STAT-dependent transcriptional activity induced by wild-type MPL or mutants of MPL. γ2A cells which are JAK2-deficient were transiently transfected with wild-type MPL (or mutants), JAK2, STAT5, and with Firefly STAT5 luciferase reporter spi-Luc and pRL-TK vector coding for Renilla luciferase. Luminescence was measured 48 hours after transfection. After transfection, medium was changed after 5 hours and replaced either with culture medium or medium supplemented with TPO (10 ng/mL). Shown are average units + SEM of 1 representative experiment performed in triplicate out of 3. **P < .001; *P < .05. (B) Number of viable Ba/F3 cells expressing wild-type MPL or different MPL mutants, in the absence of IL-3, was measured every 24 hours for 72 hours. The data are shown as the average of 2 biological replicates performed each in triplicate ± SEM. (C-D) The viability of Ba/F3 cells expressing wild-type MPL or different mutants was assessed after 72 hours in the presence of increasing concentrations of IL-3 (C) and TPO (D). Error bars represent SEM. (E-F) Ba/F3 cells expressing wild-type MPL (MPL wt-puro) but not GFP were mixed with Ba/F3 cells expressing either wild-type MPL (MPL wt-GFP), MPL Y591D (MPL Y591D-GFP), or MPL W515K (MPL W515K-GFP) and GFP in a 4:1 ratio and cultured in the presence of 1 ng/mL IL-3 (E) or 1 ng/mL TPO (F). The GFP positivity of the mixed culture was assessed every 72 hours by flow cytometric analysis. The experiment was performed in triplicate, with error bars representing SEM. SEM, standard error of the mean.