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Blood, Vol. 110, Issue 9, 3374-3383, November 1, 2007
The oncoprotein NPM-ALK of anaplastic large-cell lymphoma induces JUNB transcription via ERK1/2 and JunB translation via mTOR signaling
Blood Staber et al.
110: 3374
Supplemental materials for: Staber et al
Files in this Data Supplement:
- Document 1. Supplemental material and methods (PDF, 72.4 KB)
- Table S1. Microarray analysis (XLS, 31 KB)
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Hybridization experiments were performed using Cy 5-labeled ALCL-samples and Universal Human Reference RNA (Stratagene) as the Cy 3-labeled reference. The photomultiplier tube and the laser value for scanning were calibrated over all spots in both the Cy 3 and Cy 5 channels. The ratios were calibrated by applying normalization factors calculated from the mean intensities over all spots. Log ratios of the individual samples compared to the reference RNA are given.
- Figure S1 (JPG, 46 KB)
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Upper panel: Relative mRNA expression values of the major AP-1 factors in four ALCL cell lines. JUNB is the most highly expressed AP-1 factor, indicating its major contribution. All qRT-PCR assays showed >90% PCR efficiency. A relative comparison of AP-1 factor mRNA expression values could therefore be performed. MAC2A cJUN expression compared to MAC2A ACTB expression was used as calibrator (bars indicate SEM of triplicate experiment). Lower panel: relative ALK mRNA expression values in the same four ALCL cell lines (bars indicate SEM of triplicate experiment).
- Figure S2 (JPG, 42 KB)
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Upper panel: Relative mRNA expression values of the major AP-1 factors in wild-type, NPM-ALK expressing as well as mutant NPM-ALK kinase dead HEK-293 cells. Lower panel: Immunoblots of wild type, NPM-ALK, and kinase dead NPM-ALK expressing HEK293 cells. Lysates were analyzed for presence of Fos and JunB protein.
- Figure S3. Relative ALK mRNA expression values in vector control (VC) and NPM-ALK expressing Ba/F3 cells, as well as TonBaF.1-NPM-ALK cells inducibly expressing NPM-ALK without (unind) and 48 hours after addition of 2 µg/mL doxycycline (+dox) (bars indicate SEM of triplicate experiment) (JPG, 21.5 KB)
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- Figure S4. Western blotting of the NPM-ALK negative ALCL cell line MAC-2A and the NPM-ALK positive ALCL cell lines SR786 and Karpas-299 using phospho-specific Thr389 p70S6K antibody (JPG, 18.6 KB)
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The ALK negative ALCL cell line showed only weak staining for phospho-p70S6K indicating an inactive mTOR pathway compared to the NPM-ALK positive cell lines.
- Figure S5. Small hairpin (sh) RNA knockdown of JUNB in doxycycline induced TonBaF.1-NPM-ALK cells (JPG, 21.2 KB)
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Compared to control (scrambled sh) knockdown of JUNB did not alter caspase-3 activity (36h after addition of doxycycline; bars indicate SD of triplicate experiment).
- Figure S6. Protein stability of JunB, ERK, and β-actin upon rapamycin treatment in SR-786 (JPG, 34.4 KB)
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New protein synthesis was stopped by the addition of cycloheximide. At the indicated times points, lysates were analyzed by Western blot (WB) for the presence of JunB, ERK, and  -actin. Cells treated with cycloheximide +/- rapamycin demonstrated a continuous decrease of JunB, ERK, and -actin, indicating that rapamycin does not destabilize the JunB protein itself.
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