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Blood, Vol. 113, Issue 7, 1522-1525, February 12, 2009
Activity of the BH3 mimetic ABT-737 on polycythemia vera erythroid precursor cells
Blood Zeuner et al.
113: 1522
Supplemental materials for: Zeuner et al
Files in this Data Supplement:
- Table S1. Polycythemia vera patients (PDF, 552 KB) -
HU: Hydroxyurea
- Table S2 (PDF, 171 KB) -
Percentage of JAK2 alleles bearing the V617F mutation in CD34+ cells (CD34+ Day 0), erythroblasts obtained by culturing CD34+ cells in serum-free medium with 3 U/ml Epo (Epo 3 U/ml Day 6), erythroblasts obtained by culturing CD34+ cells in serum-free medium with 0.3 U/ml Epo (Epo 0.3 U/ml Day 6), and granulocytes. JAK2 mutational analysis was performed as described in Study Design.
- Figure S1. Epo levels do not influence erythroblast Bcl-XL expression and apoptosis in most PV cases (JPG, 29.2 KB)
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(A) Western blot analysis of Bcl-XL expression in erythroblasts derived from CD34+ cells of PV patients cultivated for 7 days in serum-free medium supplemented with 3 U/ml Epo or 0.3 U/ml Epo. The quantification of Bcl-XL levels normalized to alpha-tubulin is show in the panel below. The numbers assigned to PV samples correspond to cases described in Table S1. (B) Annexin V/7-AAD staining of erythroblasts derived from 5 healthy donors (N), 5 PV patients with high JAK2V617F allele burden (PV High) and 5 PV patients with low JAK2V617F allele burden (PV Low/Null). Bars represent the mean and SD of values obtained by treating normal erythroblasts and PV erythroblasts (both derived from CD34+ progenitors cultivated for 7 days in serum-free medium with 3 U/ml Epo) with ABT-737 at the indicated doses or with an equivalent volume of DMSO (−) in erythroid medium with 3 U/ml Epo for 48 hours. Data were analyzed by means of two-way ANOVA with Bonferroni post-tests and showed a statistically significant difference of *P<0.05 at doses of 500 nM and 1 µM between normal and JAK2V617F-high PV samples.
- Figure S2. Mitochondrial depolarization and caspase activation induced by ABT-737 (JPG, 55.7 KB)
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Normal and PV erythroid precursors were cultivated for 7 days as described in Study Design. (A) Mitochondrial membrane depolarization induced by a 24 hour treatment with 5 µM ABT-737 (ABT-737) or an equivalent volume of DMSO (−) of erythroblasts derived from three JAK2V617F¬positive PV patients (PV) and three normal controls (N). The lower panel reports the mean and SD of the percentages of TMRM-positive intact mitochondria shown in the upper panels. Mitochondrial membrane depolarization was measured by flow cytometry following incubation with 10 µg/ml TMRM (tetramethyl rhodamine methyl ester). The difference between untreated and ABT-737¬treated normal samples was not statistically significant whereas the difference between untreated and ABT-737–treated PV samples analyzed by paired t test showed a statistically significant difference of **P=0.001 between untreated and ABT-737–treated PV samples. (B) Left panel: caspase 3/7 activation in normal erythroblasts (N) and JAK2V617F-high PV erythroblasts (PV treated for 24 hours with 5 µM ABT-737, for 48 hours with 1 µM ABT-737 or with an equivalent volume of DMSO (−) as detected by a fluorogenic caspase assay (Apo-ONE Homogeneous Caspase 3/7 Assay, Promega). The experiment was performed on erythroblasts derived from 3 healthy controls and 5 PV patients. Right panel: western blot analysis of caspase-3 (casp-3) and caspase-9 (casp-9) cleavage of JAK2V617F-positive PV erythroblasts DMSO-treated (−) and treated for 48 hours with 1 µM ABT-737 or for 24 hours with 5 µM ABT-737.
- Figure S3. Effects of ABT-737 on erythroid growth, differentiation and cell cycle distribution (JPG, 55.8 KB)
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(A) Erythroblasts at day 4 of liquid culture derived from 3 JAK2V617F-high patients were plated in the presence of 20 nM ABT-737 (ABT-737) or an equivalent volume of DMSO (−). ABT-737 was either preincubated for 24 hours and washed away (ABT-737 24h) or maintained until the end of the culture (ABT-737). The difference between PV (−) and PV ABT-737 24h was not statistically significant, whereas the difference between PV (−) and PV ABT-737 showed a statistical significance of ***P<0.001 at day 10, 13 and 15 between untreated and ABT-737–treated samples according to a two-way ANOVA analysis followed by Bonferroni post-tests. (B) May-Grünwald-Giemsa (left) and Glycophorin A FITC (right) staining of day 11 and day 7 erythroblasts respectively, derived from normal (N) and JAK2V617F-positive PV erythroblasts (PV) cultured from day 4 with 20 nM ABT-737 or an equivalent volume of DMSO(−). Images were taken with a Nikon Eclipse E1000 microscope equipped with a Nikon Plan Apo 60×/1.4 NA oil immersion objective and a Nikon DXM 1200 digital camera. (C) Cell cycle distribution of normal (N) and PV erythroblasts (PV) incubated with 20 nM ABT-737 or with an equivalent volume of DMSO (−) for 24 hours (left panels) or for 4 days (right panels). Cells were incubated for 1 hour at 4°C with 0.1% Triton X-100/0.1% sodium citrate/5 µg/ml propidium iodide and analyzed by flow cytometry.
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