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Blood, Vol. 112, Issue 6, 2305-2317, September 15, 2008
Translational control of c-MYC by rapamycin promotes terminal myeloid differentiation
Blood Wall et al.
112: 2305
Supplemental materials for: Wall et al
Files in this Data Supplement:
- Table S1. Primer sequences used for QRT-PCR analysis (PDF, 61.2 KB)
- Table S2. Differential cell counts from MPRO c-MYC-ER granulocytes treated with AGN194204 (PDF, 542 KB)
- Figure 1. Rapamycin reduces expression of c-MYCT58A-ER in MPRO granulocytes (JPG, 54.2 KB)
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(A) The c-MYCT58A-ER point mutant was generated using site-directed mutagenesis and pBabe-MYCT58A-ER retrovirus was used to infect MPRO cells as previously described.43 MPRO cells expressing c-MYCT58A-ER were induced to differentiate with 10−6M AGN194204 in the presence of 200nM 4-hydroxytamoxifen (4OHT), EtOH (vehicle control) or 200nM 4OHT plus 80nM rapamycin (rapa) as indicated. Protein lysates made from cells harvested after 4 days of treatment were analysed by Western Blot for expression of c¬MYCT58A-ER protein. β-tubulin was used as a loading control.
- Figure S2. Apoptosis due to enforced expression of c-MYC is reduced by rapamycin (JPG, 104 KB)
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MPRO mycER cells were induced to differentiate with 10−6M AGN194204 in the presence of vehicle control (RXR+EtOH), 200nM 4-hydroxytamoxifen (RXR+4OHT) or 200nM 4OHT plus 80nM rapamycin (RXR+4OHT+rapa) as indicated. 2 × 105 cells were stained for 15 minutes with Annexin V-APC antibody (1/100) and 0.5ug/uL propidium iodide in Annexin binding buffer (0.01M Hepes pH7.4, 0.14M NaCl, 5uM CaCl2) then diluted to 200uL prior to collection of 10,000 events on an LSRII flow cytometer and analysis using FCS express software. (A) Representative dot plots showing the percentage of cells falling into each quadrant for the day 0 cells and AGN194204 treated cells after 2 and 4 days culture under the various conditions. (B) Percentage of Annexin V positive cells at day 0 and after 2 and 4 days of treatment with AGN194204 under the various conditions. The results are graphed as the average of 2 independent experiments. *=p<0.05.
- Figure S3. Initiation of c-Myc mRNA translation is inhibited by combination treatment with AGN194204 and rapamycin (JPG, 89.2 KB)
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(A) Polysome profiles from MPRO cells treated for 48 hours with 10−6M AGN194204 (RXR) or 10−6M AGN194204 and rapamycin (RXR+rapa). Cell lysates loaded by protein equivalence were separated 10-17% sucrose density grandients with continous A254 monitoring. (B) RNA profiles showing the 18S and 28S rRNA distribution after fractionation. 200ng denatured RNA per fraction from fractions 1-4 was processed using an Agilent Bioanlyzer 2100 and RNA nanochip (Agilent, Foster City, CA) according to the manufacturers instructions and the resulting gel images for the RXR gradient (left hand panel) and the RXR+rapa gradient (right hand panel) are shown. (C and D) c-Myc and eEF1α mRNA abundance for fractions 1 to 4 was determined by qRT-PCR in MPRO cells treated as described in (A). Values are normalised to transcript abundance in fraction 2 for RXR treated cells.
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