mTORC1 activity is required to maintain expression of c-MYC-ER in MPRO granulocytes. (A) c-MYC-ER MPRO cells were induced to differentiate with 10⁻⁶ M AGN194204 in the presence of 200 nM 4OHT, EtOH (vehicle control), or 200 nM 4OHT plus 80 nM rapamycin (rapa) as indicated. Protein lysates made from cells harvested after 2 and 4 days of treatment were analyzed by Western blotting for expression of c-MYC-ER. (B) MPRO c-MYC-ER cells were treated with 200 nM 4OHT to activate c-MYC-ER in the presence of 10⁻⁶ M AGN194204 (RXR), rapamycin (rapa), or a combination of both for 48 hours. Expression of c-MYC-ER was determined by Western blotting. For panels A and B, α-tubulin was used as a loading control. (C) Abundance of the c-MYC target genes cyclin D2, mTERT, ODC, and UBF was determined by qRT-PCR in MPRO c-MYC-ER–expressing cells after differentiation induction, in the presence of EtOH (RXR + EtOH), 4OHT (RXR + 4OHT), or 4OHT and rapamycin (RXR + 4OHT + rapa). Results were corrected for β-2-microglobulin transcript abundance and represented as fold change over day-0 controls. (D) Transcript abundance of the retinoid responsive genes Mad1 and CEBPϵ was determined by qRT-PCR as described in panel C. For panels C and D, data represent mean plus or minus SEM of 3 independent experiments.