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Blood, Vol. 113, Issue 24, 6193-6205, June 11, 2009
Identification of AML1-ETO modulators by chemical genomics
Blood Corsello et al.
113: 6193
Supplemental materials for: Corsello et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 555 KB)
- Table S1. Kasumi-1 AML1-ETO knockdown (Affymetrix U133A microarray) (PDF, 26. KB) -
Description of the samples contained in the AML1-ETO knockdown Affymetrix U133A experiments and the corresponding scan identifier.
- Table S2. U937 AML1-ETO induced samples (Affymetrix U95A microarray) (PDF, 23.1 KB) -
Description of the samples contained in the AML1-ETO inducible Affymetrix U95A experiments and the corresponding scan identifier.
- Table S3. AML1-ETO marker genes (PDF, 90.7 KB) -
List of AML1-ETO marker gene names, RefSeq numbers, and probe sequences.
- Table S4. Myeloid differentiation marker genes (PDF, 74.9 KB) -
List of myeloid differentiation signature marker gene names, RefSeq numbers, and probe sequences.
- Table S5. Effects of methylprednisolone and methotrexate on AML cell line viability (PDF, 19 KB) -
Day 3 and day 5 IC50 values in AML cell lines for methylprednisolone and methotrexate.
- Figure S1. Performance of siRNA directed against AML1-ETO (JPG, 149 KB)
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(A) Kasumi-1 immunoblot with ETO antibody demonstrated loss of AML1-ETO protein expression 96 hours following transient transfection by Amaxa with siRNA directed against the AML1-ETO breakpoint versus a luciferase directed control. ETO expression remains stable. β-Actin was used as a loading control. (B) Real-time PCR with AML1-ETO specific primers reveals loss of AML1-ETO expression at 24 hours with siRNA directed against the breakpoint. Full recovery of transcript expression occurs by 5 days post-transfection. Experiments were performed in replicate of four.
- Figure S2. AML1-ETO signature is modulated by dexamethasone and pyrimethamine (JPG, 84.4 KB)
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Weighted summed score for the AML1-ETO signature upon Kasumi-1 treatment with DMSO, dexamethasone, or pyrimethamine for 72 hours. There were 32 replicates for the untreated controls and 4 replicates for each of the drug concentrations and DMSO controls. The DMSO concentration corresponds to the maximal compound hit DMSO content. Error bars depict the standard deviation of the measurements.
- Figure S3. Dexamethasone and pyrimethamine induce increase in differentiation in AML1-ETO positive cell lines (JPG, 321 KB)
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Each condition was evaluated in duplicate. CD11b/CD14 cell surface staining of (A) Kasumi-1 and (B) SKNO-1 cells at 72 hours. Treatment with pyrimethamine induced upregulation of CD14 and CD11b while dexamethasone induced a subtle upregulation of CD14 and double staining in the Kasumi-1 cells.
- Figure S4. Dexamethasone and pyrimethamine induce apoptosis in AML1-ETO positive cell lines (JPG, 749 KB)
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Annexin V/propidium iodide staining of (A) Kasumi-1 cells and (B) SKNO-1 cells 72 hours following treatment with either dexamethasone or pyrimethamine. Both compounds increased Annexin V cell surface staining in a dose-responsive manner. Each condition was evaluated in duplicate.
- Figure S5. Methylprednisolone decreases BCL2 protein in Kasumi-1 cells (JPG, 272 KB)
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Kasumi-1 cells were treated with either DMSO, methylprednisolone, or methotrexate at four increasing doses and expression of BAX, BIM, and BCL2 evaluated at 24 and 48 hours by immunoblotting.
- Figure S6. Effects of AML1-ETO expression in U937 on methotrexate and methylprednisolone induced alterations of viability and differentiation (JPG, 272 KB)
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U937 cells conditionally expressing AML1-ETO under the control of the tetracycline promoter were grown in the presence (AML1-ETO off) or absence of tetracycline (AML1-ETO on) for three days. (A) Immunoblotting was performed for AML1-ETO with an anti-ETO antibody. (B) Cells were then treated with methotrexate and methylprednisolone in a 2-fold dilution series for three additional days and viability measured by an ATP-based assay in replicate of five. AML1-ETO expression sensitized cells to the effects of both compounds on viability. (C) Cells were treated with methotrexate or methylprednisolone in a 2-fold dilution series for three more days and a 32-gene myeloid differentiation signature (Weighted summed score) evaluated by GE- HTS. Each condition was evaluated in replicate of 10. AML1-ETO expression did not sensitize the cells to the effects of compounds on myeloid maturation.
Additional supplemental figures can be found here.
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