mTORC1 activity is required for c-MYC–mediated cell growth in granulocytes. (A) MPRO c-MYC-ER cells were induced to differentiate with 10⁻⁶ M AGN194204 and treated with EtOH (RXR + EtOH), 200 nM 4OHT (RXR + EtOH), or 200 nM 4OHT and 80 nM rapamycin (RXR +4OHT + rapa). The mean cell volume (MCV) was determined at the indicated time points by the Coulter method. (B) Protein synthesis was determined by measuring incorporation of ³⁵S methionine into total cellular protein in c-MYC-ER MPRO cells treated as indicated as described for panel A. Results at day 2 and day 4 are represented as the percentage counts per minute relative to undifferentiated (day 0) cells. (C) c-MYC-ER MPRO cells (10⁷) treated as described in panel A were lysed in 1% SDS lysis buffer containing protease inhibitors. Total protein per cell was determined using a modified Lowry assay. Results at day 2 and day 4 are represented as fold change relative to values for undifferentiated (day 0) cells. (D) Abundance of the unprocessed 45S rRNA transcript was determined by qRT-PCR using primers directed against the 5′ ETS region, corrected for β-2-microglobulin transcript abundance and represented as fold change over day 0 for MPRO c-MYC-ER cells induced to differentiate under the conditions described in panel A. (E) The percentage of cells in S phase was determined by FACS analysis of BrdU-positive cells for MPRO c-MYC-ER cells under the conditions described in panel A. For all sections, the data shown are the mean plus or minus SEM of 3 independent experiments. *P < .05, Student t test.