Induction of Bim is regulated by JAK2 kinase activity. (A) Apoptosis induced by Epo withdrawal. Ba/F3-EpoR cells (parental), Ba/F3-EpoR-wtJAK2, and Ba/F3-EpoR-V617F cells were washed 3 times with RPMI only and resuspended in RPMI supplemented with 10% fetal calf serum in the absence of Epo for the indicated hours. Cells were harvested, and apoptosis was measured for incorporation of propidium iodide by flow cytometry. Data are mean ± SD of 3 independent experiments. JAK2 WT versus JAK2 V617F: *P < .05, ***P < .001. (B) Western blot analysis of total-Bim and phospho-Bim_. Ba/F3 stable cells was cultured in the presence of Epo for 0, 12, and 24 hours. (C) Apoptosis induced by JAK2 V617F inhibition. Ba/F3-EpoR-V617F cells were treated with either 3μM JAK inhibitor I or 0.1% DMSO (control) for 0, 24, 48, and 72 hours in RPMI supplemented with 10% fetal calf serum. Cells were harvested and apoptosis was assessed by flow cytometry. Data are mean ± SD of 3 independent experiments. Control versus JAK inhibitor I: *P < .05, **P < .01. (D) Induction of Bim by JAK2 V617F inhibition. Ba/F3-EpoR-V617F cells were treated with either 3μM JAK inhibitor I or 0.1% DMSO (control) for 0, 6, 12, and 24 hours. Cells were harvested and analyzed by Western blot.