Total c-Myc mRNA and c-MYC protein stability do not change in MPRO granulocytes treated with rapamycin. (A) Abundance of c-MYC-ER mRNA in c-MYC-ER MPRO cells induced to differentiate with 10−6 M AGN194204 and treated with 200 nM 4-hydroxytamoxifen (RXR + 4OHT) or 200 nM 4OHT and 80 nM rapamycin (RXR + 4OHT + rapa) was determined by qRT-PCR. Results were corrected for β-2-microglobulin transcript abundance and represented as fold change over untreated (day 0) cells. (B) Abundance of c-MYC mRNA was determined by qRT-PCR in MPRO cells treated with EtOH, 10−6 M AGN194204 (RXR), 80 nM rapamycin (rapa), or 10−6 M AGN194204 and 80 nM rapamycin (RXR + rapa). Results were corrected for β-2-microglobulin transcript abundance and represented as fold change relative to EtOH control. For panels A and B, data shown are the mean plus or minus SEM of 3 independent experiments. (C) c-MYC-ER MPRO cells were treated with 200 nM 4OHT and either EtOH or 80 nM rapamycin. Cycloheximide (10 μg/mL) was added to inhibit new protein synthesis and cells were harvested after 0, 30, 60, and 120 minutes. Protein lysates were analyzed for c-MYC-ER expression by Western blotting and α-tubulin was used as a loading control. To determine protein turnover, band densitometry was performed using ImageJ software. c-MYC-ER band density was corrected for α-tubulin and represented as fold change over time 0. Calculated band density normalized to time 0 is shown under the Western blot. (D) Cycloheximide (10 μg/mL) was added to MPRO cells treated with EtOH or 80 nM rapamycin. Lysates were generated from cells harvested at 0, 20, 40, 60, and 120 minutes. Endogenous c-MYC and α-tubulin levels were determined by Western blotting and quantitated by ImageJ software to determine protein turnover as described in panel C. Calculated band density for the gel pictured is shown under the Western blot. Quantitation in the right hand panel is the mean of 2 independent experiments.
