JAK2^ex13InDel confers cytokine-independent growth to Ba/F3 cells. (A) Parental Ba/F3 cells were transduced with human JAK2-GFP retroviral constructs for expression of JAK2^WT, JAK2^V617F, or JAK2^ex13InDel and cultured in WEHI conditioned medium as a source of IL-3. At 48 hours after transduction, IL-3 withdrawal led to rapid selection of JAK2^ex13InDel-expressing cells (n = 3). (B) Parental Ba/F3 cells and GFP-sorted Ba/F3 cells expressing JAK2 constructs were plated at equal numbers and grown with or without IL-3. All cells proliferated in the presence of IL-3 (not shown), while only those cells containing JAK2^ex13InDel exhibited exponential growth in the absence of IL-3 (n = 3). (C) Parental Ba/F3 cells and JAK2^WT- and JAK2^V617F-expressing Ba/F3 cells display growth sensitivity to varying concentrations of IL-3, while growth of Ba/F3 JAK2^ex13InDel cells is not affected by IL-3 concentration (n = 3). (D) To assess the effect of JAK2 inhibition on Ba/F3 cells, we performed cell proliferation experiments with JAK inhibitors. The IC₅₀ of momelotinib was increased 2-fold in JAK2^ex13InDel Ba/F3 cells (n = 3). The IC₅₀ of ruxolitinib was increased 10-fold in JAK2^ex13InDel Ba/F3 cells compared with controls and JAK2^V617F Ba/F3 cells.