![Figure 1. PV erythroblasts overexpress Bcl-2 and Bcl-XL and are sensitive to ABT-737–induced apoptosis. CD34+ hematopoietic progenitors isolated from healthy donors and patients with PV were grown in liquid culture for 7 days in medium containing 3 U/mL erythropoietin (for normal HPCs) or 0.3 U/mL erythropoietin (for PV HPCs) as described in “Study design.” The numbers assigned to PV samples correspond to patients described in Table S1. (A) Western blot analysis of Bcl-2, Bcl-XL, and Mcl-1 levels in erythroblasts of healthy controls (N) and PV patients (PV). The right panel shows the quantification of protein levels normalized to beta-actin. (B) Levels of Bcl-2 and Bcl-XL expression in erythroblasts derived from 15 healthy donors or 18 patients with PV with different JAK2V617F allele burdens. Values correspond to the quantification of Western blot bands normalized to beta-actin. Data were analyzed by means of nonparametric Kruskal-Wallis test and the Dunn post-test to compare each group (***P < .001 [Bcl-2]; *P < .05 [Bcl-XL] between normal and JAK2V617 high erythroblasts). (C) Cell death induced by treatment of normal and PV erythroblasts for 16 hours with 75 μM cytosine arabinoside (AraC) or 150 μM hydroxyurea as evaluated by ethidium bromide/acridine orange staining and fluorescence microscopy. Mann-Whitney nonparametric test showed statistical significance (**P = .006 [AraC]; **P = .002 [hydroxyurea] between normal and JAK2V617F-medium/high erythroblasts). (D) Annexin V/7-AAD staining of erythroblasts derived from 5 healthy donors (N), 5 patients with PV with low JAK2V617F allele burden (PV Low/Null), and 5 patients with PV with high JAK2V617F allele burden (PV High). Bars represent the mean and SD of values obtained by treating normal and PV erythroblasts with ABT-737 at the indicated doses or with an equivalent volume of DMSO (−) for 48 hours. The left panel shows a representative experiment from each of the categories analyzed, with numbers indicating the total percentage of Annexin V+ cells. Data were analyzed by means of 2-way analysis of variance (ANOVA) with Bonferroni post-tests and showed a statistically significant difference (*P < .05 at doses of 500 nM and 1 μM between normal and JAK2V617F-high PV samples). Experiments shown in panels C and D were conducted in erythroid medium containing 0.3 U/mL Epo for both normal and PV erythroblasts.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/113/7/10.1182_blood-2008-03-143321/4/zh80090931510001.jpeg?Expires=1724264745&Signature=qfkORzGqF-8ILFsEVTZY3zVS1Gfl9-29rtWCK2rrdiV3qfiDO43YvwjSmgAo204ziOmt-TFPGrsJf406VUJwWc5YLXjxLMwTuCqCa6OnWyVmcjA5nl5Btt2bS924eApdo598traCY9EqdfwNEGvaEZicmMZqeAWqUC5Xv8FLLfyRIyma8o~svOb9~oPWHip6dXgU-S3LHR~AompkaQUPe02VGnn8rII0vlC9~XffZmy-YjEIhzKgW19pPUWIWNAOHkBlSjaFkvx8oCDRDfGWS-v5LbffeDY7NvPBQzFzncQvdxhrvRHAeLUU2tAlH3Q0RoQNFWdrsH515Q~oqFYpgQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
PV erythroblasts overexpress Bcl-2 and Bcl-XL and are sensitive to ABT-737–induced apoptosis. CD34+ hematopoietic progenitors isolated from healthy donors and patients with PV were grown in liquid culture for 7 days in medium containing 3 U/mL erythropoietin (for normal HPCs) or 0.3 U/mL erythropoietin (for PV HPCs) as described in “Study design.” The numbers assigned to PV samples correspond to patients described in Table S1. (A) Western blot analysis of Bcl-2, Bcl-XL, and Mcl-1 levels in erythroblasts of healthy controls (N) and PV patients (PV). The right panel shows the quantification of protein levels normalized to beta-actin. (B) Levels of Bcl-2 and Bcl-XL expression in erythroblasts derived from 15 healthy donors or 18 patients with PV with different JAK2V617F allele burdens. Values correspond to the quantification of Western blot bands normalized to beta-actin. Data were analyzed by means of nonparametric Kruskal-Wallis test and the Dunn post-test to compare each group (***P < .001 [Bcl-2]; *P < .05 [Bcl-XL] between normal and JAK2V617 high erythroblasts). (C) Cell death induced by treatment of normal and PV erythroblasts for 16 hours with 75 μM cytosine arabinoside (AraC) or 150 μM hydroxyurea as evaluated by ethidium bromide/acridine orange staining and fluorescence microscopy. Mann-Whitney nonparametric test showed statistical significance (**P = .006 [AraC]; **P = .002 [hydroxyurea] between normal and JAK2V617F-medium/high erythroblasts). (D) Annexin V/7-AAD staining of erythroblasts derived from 5 healthy donors (N), 5 patients with PV with low JAK2V617F allele burden (PV Low/Null), and 5 patients with PV with high JAK2V617F allele burden (PV High). Bars represent the mean and SD of values obtained by treating normal and PV erythroblasts with ABT-737 at the indicated doses or with an equivalent volume of DMSO (−) for 48 hours. The left panel shows a representative experiment from each of the categories analyzed, with numbers indicating the total percentage of Annexin V+ cells. Data were analyzed by means of 2-way analysis of variance (ANOVA) with Bonferroni post-tests and showed a statistically significant difference (*P < .05 at doses of 500 nM and 1 μM between normal and JAK2V617F-high PV samples). Experiments shown in panels C and D were conducted in erythroid medium containing 0.3 U/mL Epo for both normal and PV erythroblasts.
