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Blood, Vol. 112, Issue 8, 3403-3411, October 15, 2008
Combined functional and molecular analysis of tumor cell signaling defines 2 distinct myeloma subgroups: Akt-dependent and Akt-independent multiple myeloma
Blood Zöllinger et al.
112: 3403
Supplemental materials for: Zollinger et al
Files in this Data Supplement:
- Table S1. Characteristics of primary MM cells/MM patients (PDF, 51.7 KB) -
Viability: MM cells cocultured with BMSCs measured by annexin V-FITC/PI staining after 5 d of Akti-1/2 (10 µM) treatment. Percentage survival is calculated relative to DMSO-treated controls. IHC: phospho-Akt staining. Flow cytometry: extent of MFI-change of DMSO versus Akti-1/2 treated samples determined by phospho-Akt-specific flow cytometry. t(4;14): translocation t(4;14). PTEN del: deletion of PTEN. del13q: deletion of 13q. Additionally, information on the sex, age, stage of disease (stage), immunoglobulin type (Ig type) and number of previous therapies (prev therapies) is displayed. Due to limited material, there does not exist a complete data set for each patient. wt=wildtype; t=translocation; del=deletion; f=female; m=male. For information on FISH analysis see Leich E, Haralambieva E, Zettl A, et al. Tissue microarray-based screening for chromosomal breakpoints affecting the T-cell receptor gene loci in mature T-cell lymphomas. J Pathol. 2007;213:99-105. Probes were purchased from Abbot GmbH & Company KG (Wiesbaden, Germany): LSI IGH/FGFR3 Dual Color/ Dual Fusion DNA Probe (#32-191023), LSI PTEN/CEP10 Dual Color DNA Probe (#32-231010), LSI 13(13q14) Spectrum Green DNA Probe (#32-192018). Grey highlighting marks patient samples counted as resistant/resilient to treatment with Akti-1/2.
- Figure S1. Transfection and enrichment of AMO-1 and MM.1S cells (JPG, 469 KB)
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MM cells were electroporated with pSUPER siRNA expression constructs and an expression plasmid for enhanced green fluorescent protein (EGFP). Left vertical row: cultures one day after electroporation as they appear after PI-staining and flow cytometry for EGFP and PI. The lower left quadrant marks viable cells, the upper left quadrant dead cells and both quadrants to the right the live transfected (EGFP-positive) fraction. The EGFP-signal in very bright live cells is also registered in the PI-channel. Middle vertical row: The same samples after sorting for bright green cells. Right vertical row: The same samples after five additional days in culture. Visible are the decline of EGFP-intensity (indicative of expression plasmid depletion through dilution (i.e. proliferation) or degradation), in all samples except Akt1 + Akt2 depleted MM.1S cells, which lose the signal alltogether and turn PI-positive.
- Figure S2. Establishment of phospho-Akt (Ser473)-specific flow cytometry with AMO-1 and MM.1S cells (JPG, 86.8 KB)
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For subsequent experiments with primary MM cells phospho-Akt-specific flow cytometry was established in MM cell lines. DMSO-treated AMO-1 cells do not show different phospho-Akt signaling from cells treated with Akti-1/2 (10 µM), or from a control stained with the fluorochrome-coupled secondary antibody only. In contrast, DMSO-treated MM.1S cells show higher mean fluorescence intensity (MFI) than Akti-1/2 treated cells, which reflects the presence of phosphorylated protein. Of note, Akti-1/2 does not change the MFI of AMO-1 or of MM.1S cells indicating that the drug has no influence on the autofluorescence of the cells.
- Figure S3. Western analysis showing the specificity of Akt isoform-specific antibodies (JPG, 73.7 KB)
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Recombinant Akt isoforms were obtained from CST (Akt1 kinase (#7500), Akt2 kinase (#7503), Akt3 kinase (#7506)) and probed with the antibodies against Akt1, Akt2, Akt3 and pan Akt mentioned in the Material and Methods section.
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