Efficacy of CYT387 against JAK2-dependent cell lines in vitro. (A) Chemical structure of CYT387. (B) Ba/F3-EpoR parental (cultured in media supplemented with 3 U/mL erythropoietin) or Ba/F3-EpoR-JAK2^V617F cells (cultured in the absence of exogenous cytokines) were plated in 96-well plates over a dose gradient of CYT387 for 3 days at which point cell viability was measured with an MTS tetrazolium salt assay. Values represent mean ± SEM. n = 3; *P < .05. (C) Baf3-EpoR-JAK2^V617F cells were plated in graded concentrations of CYT387 for 1 day (immunoblot) or 2 days (trypan blue exclusion) at which point cell death was measured by trypan blue exclusion and apoptosis was determined by immunoblot with antibodies specific for cleaved caspase 3 and tubulin. Values represent mean ± SEM. n = 3; *P < .05. (D) Ba/F3-EpoR-JAK2^WT or Ba/F3-EpoR-JAK2^V617F cells were serum-starved then plated in 6-well plates over a dose gradient of CYT387 for 16 hours at which point cells were lysed and whole-cell extracts were subjected to immunoblot analysis using antibodies specific for total or phospho-JAK2, total or phospho-ERK1/2, total or phospho-STAT5, or β-actin.