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Blood, Vol. 113, Issue 8, 1710-1722, February 19, 2009
Small molecule XIAP inhibitors cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells and overcome Bcl-2–mediated resistance
Blood Fakler et al.
113: 1710
Supplemental materials for: Fakler et al
Peripheral blood lymphocytes (PBLs) were isolated from buffy coats of healthy donors by ficoll separation (Biochrom) and cultured in RPMI1640 medium supplemented with 30 units/ml human recombinant interleukin-2 (Biochrom). Upon purification, PBLs were treated with 2.5 µl/ml phytohemagglutinin (Sigma) for 24h and allowed to proliferate for 6 days before the start of the experiment. Jurkat cells transfected with XIAP were kindly provided by Dr. C. Duckett (Ann Arbor, MI).45
Files in this Data Supplement:
- Figure S1. Inhibition of TRAIL-induced apoptosis by XIAP (JPG, 58.6 KB)
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(A) Effect of XIAP overexpression on TRAIL-induced apoptosis. Jurkat cells transfected with XIAP were analyzed for XIAP expression by western blotting (upper panel) and were treated for 72h with indicated concentrations of TRAIL and/or 10 nM XIAP inhibitors, control compound or DMSO (lower panels). Apoptosis was determined by forwardside scatter analysis and flow cytometry. Mean and SD of 3 experiments each performed in triplicate are shown. (B) Effect of TRAIL on XIAP expression. Jurkat cells were treated for indicated times with 0.4 ng/ml TRAIL and analyzed for XIAP expression by western blotting.
- Figure S2. Effect of caspase-3 inhibition on caspase-8 activation upon treatment with XIAP inhibitor and TRAIL (JPG, 76.4 KB)
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Jurkat cells were treated for 24h with 0.4 ng/ml TRAIL and 10 nM XIAP inhibitor 2, control compound or DMSO in the absence or presence of 50 µM zDEVD.fmk or DMSO. Caspase activation was analyzed by Western blotting. Arrowheads indicate caspase cleavage fragments.
- Figure S3. Effect of Bcl-2 overexpression on XIAP inhibitor-induced apoptosis (JPG, 49.8 KB)
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Jurkat cells transfected with empty vector (Neo) or Bcl-2 were treated for 72h with indicated concentrations of XIAP inhibitors, control compound or DMSO. Apoptosis was determined by forwardside scatter analysis and flow cytometry. Mean and SD of 3 experiments each performed in triplicate are shown.
- Figure S4. XIAP inhibitors do not reverse the lack of toxicity of TRAIL on normal lymphocytes (JPG, 120 KB)
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(A–B) Effect of XIAP inhibitors on apoptosis of PBLs. Unstimulated (A) and PHA-stimulated (B) PBLs were left untreated (white bars) or were treated for 72h with indicated concentrations of XIAP inhibitors, control compound or DMSO. Apoptosis was determined by forwardside scatter analysis and flow cytometry. Mean and SD of 3 experiments each performed in triplicate are shown, each experiment was performed with PBLs of an individual donor. (C–F) Effect of XIAP inhibitors on TRAIL- or CD95-induced apoptosis in PBLs. Unstimulated (C, E) and PHA-stimulated (D, F) PBLs were treated for 72h with indicated concentrations of TRAIL (C–D) or anti-CD95 agonistic antibody (E–F) in the absence (white bars) or presence of 10 nM XIAP inhibitors, control compound or DMSO. Apoptosis was determined by forwardside scatter analysis and flow cytometry. Mean and SD of 3 experiments each performed in triplicate are shown, each experiment was performed with PBLs of an individual donor. (G), Expression of TRAIL receptors and CD95 on PBLs. Surface expression of TRAIL receptors TRAIL-R1 to -R4 and CD95 on unstimulated and PHA-stimulated PBLs was determined using fluorescence-conjugated antibodies and flow cytometry (dotted line: cells stained with isotype control, thick line: cells stained with anti–TRAIL-R1 to -R4 or anti-CD95 antibodies). HL-60 cells were used as positive control for staining of TRAIL-R1, -R3, and -R4 (data not shown). Fluorescence intensity (x-axis) is blotted against counts (y-axis). A representative experiment of 3 independent experiments is shown. (H), Expression of IAPs and IAP antagonists in PBLs. Expression of XIAP, cIAP1, cIAP2, survivin, livin, Smac, Omi, and β-actin was assessed in Jurkat leukemia cells and in unstimulated and PHA-stimulated PBLs by Western blotting. Reh cells were used as positive control for livin expression.
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