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Blood, Vol. 111, Issue 8, 4338-4347, April 15, 2008
Loss of TLE1 and TLE4 from the del(9q) commonly deleted region in AML cooperates with AML1-ETO to affect myeloid cell proliferation and survival
Blood Dayyani et al.
111: 4338
Supplemental materials for: Dayyani et al
Files in this Data Supplement:
- Table S1. RT-PCR primers for del(9q) CDR genes (JPG, 123 KB)
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- Figure S1. Induction of AML1-ETO leads to cell cycle arrest and cell death in U937T-A/E cells (JPG, 60.3 KB)
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(A) Time-dependent induction of AML1-ETO after tetracycline withdrawal as analyzed by RT-PCR in U937T-A/E cells. The quality of the RNA was assessed by RT-PCR for beta-2-microglobulin (B2M). Note by day 11 most of the cells were dead as reflected by a low B2M band. (B) The cell cycle distribution of U937T-A/E cells was analyzed at the indicated times after tetracycline withdrawal. By 9 days most cells are dead with a prominent subG1 peak.
- Figure S2. Knockdown of TLE levels inhibits the apoptosis and cell death induced by AML1-ETO expression in U937T-A/E cells (JPG, 101 KB)
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U937T-A/E cells were infected with lentivirus containing – (A), a control shRNA (TLE4scr3); (B and C), shRNAs directed against both TLE1 and TLE4 (TLE1/4si2, TLE1/4si3); (D), shRNAs specific for TLE1 (TLE1si1) or (E), TLE4 (TLE4si2) 2 days prior to tetracycline withdrawal and AML1-ETO induction. In each case greater than 95% of cells were infected as judged by GFP expression (not shown). Cells were cultured an additional 6 days then stained with Annexin-V and 7-AAD to detect cells undergoing early apoptosis (Annexin-V positive, 7-AAD negative), or dead cells/cells in late apoptosis (Annexin-V positive, 7-AAD positive). Two independent experiments were performed and representative plots are shown (A-E). The percentage of cells in each quadrant is indicated. Results are summarized in panel (F) expressed as the mean ± SEM. (*) indicates p<0.05.
- Figure S3. Induction of apoptosis in Kasumi cells following TLE1 or TLE4 expression (JPG, 91.4 KB)
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Kasumi cells were infected with retroviral supernatants containing full-length TLE1 or TLE4 cDNA or empty MSCV-IRES-GFP vector as a control. Two days after transduction, GFP positive cells were sorted by FACS, and after 4 additional days in culture cells were analyzed for apoptosis. The percentage of cells in each quadrant is indicated. Expression of either TLE1 or TLE4 increased the percentage of early apoptotic cells (Annexin-V+/7AAD−) and the percentage of late apoptotic and dead cells (Annexin-V+/7AAD+).
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