Figure 1. Ras-mediated transactivation of the human MCSF receptor promoter using either pRL-0 or pRL-CMV as an internal control plasmid.(A) The MCSF receptor promoter in pXP2 and the CMV promoter in pRL-0 are both activated by oncogenic ras, whereas the promoterless vector pRL-0 is not. For transactivation assays, 2× 105 CV1 cells (CCL-70; ATCC, Rockville, MD, USA) were plated in 4 mL of Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 60-mm tissue culture plates 18 h before transfection. Cells were transfected by the calcium phosphate method as previously described (4, 14) with:(i) the reporter plasmid: 10 µg of the promoterless firefly luciferase vector pXP2; 10 µg of the 159-bp human MCSF receptor promoter (pMCSFR)(-88 to+ 71 with respect to the major monocytic transcription start site) in pXP2; 0.002 µg of the CMV promoter in the firefly luciferase vector pXP2; 0.002 µg of an internal control plasmid using a CMV promoter subcloned in pRL-0 (Renilla luciferase gene driven by the CMV promoter)(11) or 0.2 or 0.8 µg of the pRL-0 vector for Renilla luciferase, which lacks eukaryotic promoter and enhancer elements upstream of Renilla luciferase;(ii) 1 µg of the expression vector: oncogenic pMT3-H-Ras (L61) or the empty expression vector; and (iii) sheared salmon sperm DNA to a total of 15 µg DNA per plate. The medium was changed after 16 h, and firefly luciferase activities for pXP2 alone and pM-CSFR or pCMV in pXP2 and Renilla luciferase activities for pCMV in pRL-0 and pRL-0 alone were determined 40 h after the initiation of the transfection protocol with the Dual-Luciferase™ Reporter Assay System (Promega). Results were normalized for extract protein content and are given as mean plus standard error of the mean (SEM) of at least 6 independent experiments.(B) Using pRL-0 as an internal control plasmid results in an accurate calibration of the 4-fold transactivation of the MCSF receptor promoter by activated ras, whereas pRL-CMV can not be used as an internal control. Firefly luciferase activities of pMCSFR in pXP2 were normalized to the Renilla luciferase values of either pCMV in pRL-0, 0.2 µg pRL-0 or 0.8 µg pRL-0 (data from Panel A).