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Blood, Vol. 111, Issue 4, 2269-2279, February 15, 2008
A critical role for Lyn in acute myeloid leukemia
Blood Dos Santos et al.
111: 2269
Supplemental materials for: Dos Santos et al
Files in this Data Supplement:
- Figure S1. Analysis of the global protein tyrosine phosphorylation in AML subtypes (JPG, 55.2 KB)
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The level of protein tyrosine phosphorylation in normal unstimulated CD34+ HPCs and in AML samples according to their cytogenetic or molecular background, was monitored by Western Blot with the anti-phosphotyrosine antibody 4G10. The membranes were stripped and reprobed with an anti- -actin antibody as loading control.
- Figure S2. SFK family members in AML: expression and activity (JPG, 49 KB)
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(A) The levels of Fyn, Lck, Hck, Fgr and Lyn in normal CD34+ HPCs and AML samples, were analyzed by Western Blot using specific antibodies (patients # 7; 15; 19; 22; 25; 30-33; 35-37). The level of -actin was analyzed as loading control (lower panel). For Lck, human platelet homogenates were used as a positive control (not shown). Results shown are representative of the different patients analyzed. (B) Lyn is a major tyrosine phosphorylated protein in AML. Lyn was depleted from KG1 and U937 cell lysates by immunoprecipitation. Lyn-depleted lysates (Lyn IP) were then analyzed by Western Blot with the antiphosphotyrosine 4G10 antibody. As a control total extract (TE) and the non-depleted lysates (Ctrl IP) were also analyzed. (C) Lyn in vitro kinase assay. Lyn was immunopurified from 5 × 106 Peripheral Blood Lymphocytes (PBL), KG1 and AML patient cells as describe in Material and Methods, washed twice in lysis buffer and once with kinase buffer containing 50 mM Tris/HCl pH 7,4, 2,5 mM MnCl2 and 5 mM MgCl2). The beads were resuspended in 40 µL of kinase buffer containing 10 µCi of  -32P ATP (Perkin-Elmer) with cold ATP and incubated at 37°C for 30 minutes under shaking. Reactions were terminated by addition of 10 µL of 5X Laemli buffer. The samples were submitted to a 8% SDS-PAGE gel and Lyn autophosphorylation was evaluated by PhopshorImager analysis.
- Figure S3. Effect of PP2 on KG1 cell cycle and proliferation (JPG, 36.9 KB)
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(A) KG1 cells were grown in clonogenic assays in the presence of increasing doses of PP2. Results are presented as percentage of CFU-L for each PP2 concentration relative to untreated cells and are mean ± SEM of three independent experiments performed in duplicate. (B) KG1 cells were cultured in liquid medium alone or with increasing doses of PP2 for 4 days. Cell viability was quantified by MTT assay. Results are mean ± SEM of three independent experiments performed in triplicate. (C) KG1 cells were cultured in liquid culture alone or with increasing doses of PP2 for 24 hours then processed for cell cycle analysis. Briefly, cells were washed in phosphate-buffered saline (PBS; NaCl 7.6 g/L; Na2HPO4 0.7 g/L; KH2PO4 0.2 g/L) with 5.5 mM glucose at 4°C and fixed in 70% ethanol overnight at 4°C. Cells were then resuspended in 1 mL PBS containing 50 µg/mL propidium iodide and 100 U/mL RNAse A and incubated for 30 minutes at room temperature. The DNA content was monitored by flow cytometry (EPICS XL-MCL; Beckman Coulter, Villepinte, France). Percentages of cells in G0/G1, S, G2/M phases are indicated in each plot. Results shown are representative of three experiments.
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