JAK2V617F requires tyrosine 201 for cytokine-independent proliferation and transformation of Ba/F3-EpoR cells. Ba/F3-EpoR cells stably expressing JAK2WT, JAK2V617F, and JAK2V617F/Y201F were used. (A) JAK2 protein expression was assessed in these cell lines by Western blot. (B) Cytokine-independent cell proliferation was determined over 3 days using the WST assay. (C) Total numbers of viable cells were counted over 5 days in the absence of cytokine by trypan blue exclusion. In both cases, JAK2V617F-induced cytokine-independent cell proliferation was significantly inhibited by the Y201F mutation. Results shown are representative of 3 independent experiments. Data are mean ± SEM. (D) Flow cytometric analysis shows a marked increase in apoptosis (annexin V⁺) in Ba/F3-EpoR cells expressing JAK2V617F/Y201F compared with those expressing JAK2V617F 2 days after cytokine withdrawal. Representative dot plots from 3 independent experiments are shown. (E) Percentages of annexin V⁺ cells from 3 independent experiments are shown in bar graphs as mean ± SEM. Noticeably, the population undergoing apoptosis (annexin V⁺) in Ba/F3-EpoR cells expressing JAK2V617F/Y201F was significantly higher than those expressing JAK2V617F and identical to those expressing JAK2WT. (F) The Y201F mutation remarkably inhibited the transformation induced by JAK2V617F. Ba/F3-EpoR cells expressing different JAK2 mutants (2.5 × 10² cells/dish) were plated in duplicate in methylcellulose medium without any cytokine. Colonies were counted after 5 days. Data are mean ± SEM from 3 independent experiments. **P < .005 (1-way ANOVA).