ABT-737 enhances the effect of JAK inhibitor I on HEL cells and primary CD34⁺ cells isolated from PV patients. (A) Annexin V assay. HEL and SET-2 cells were treated with increased doses of JAK inhibitor I in the absence (open bars) or presence (black bars) of 0.3μM of ABT-737 for 24 hours. Then, cells were harvested and apoptosis was measured by flow cytometry. Data are mean ± SD of annexin V⁺ cells. Error bars represent SD (n = 3). *P < .05. **P < .01. ***P < .001. (B) Colony formation assay of primary CD34⁺ cells in the presence of Epo. CD34⁺ cells were isolated from JAK2 V617F⁺ PV patients (top panel, black bars) and healthy volunteers (bottom panel, open bars). Cells were seeded in methylcellulose medium containing Epo (H4434) and various concentrations of ABT-737 (0, 0.3, and 1μM) and JAK inhibitor I (0, 0.1, and 0.3μM), where indicated. Erythroid colonies were scored after 14 days. Data are the mean ± SD of erythroid colony numbers expressed as a percentage of DMSO-treated cultures. Error bars represent SD of PV patients (n = 5) and healthy controls (n = 5). *P < .05. **P < .01. ***P < .001. (C) Assessment of JAK2 V617F mutation frequency in colony-forming cells. CD34⁺ cells were isolated from 7 JAK2 V617F⁺ PV patients. Cells were seeded in methylcellulose medium in the presence of Epo (H4434) and/or 0.3μM of ABT-737 and 0.3μM of JAK inhibitor I, where indicated. After 14 days of culture, individual erythroid colonies were isolated from each plate and genomic DNA was extracted. Quantitative real-time PCR was used to detect the presence of JAK2 V617F. Black bars represent the percentage of JAK2 V617F⁺ colonies; open bars, percentage of JAK2 WT colonies in cultured cells as detected in 4 to 8 genomic DNA samples/condition from informative PCRs (samples showing signals detectable in the JAK2 V617F and/or the JAK2 WT reaction were considered informative). (D) Colony formation assay in the absence of Epo. CD34⁺ cells were isolated from JAK2 V617F⁺ PV patients. Cells were seeded in methylcellulose medium lacking Epo (H4534) in the presence or absence of indicated concentrations of ABT-737 and/or JAK inhibitor I. Independent EECs were counted based on benzidine staining (supplemental Figure 5). Data are mean ± SD of EEC colony numbers expressed as percentage of DMSO-treated cultures. Error bars represent SD (n = 5). *P < .05. **P < .01. ***P < .001.