FIG. 7.

Influence of SNURF overexpression on AR-dependent transactivation. (A) SNURF enhances AR-dependent and basal transcription from the rat probasin promoter. COS-1 cells were transfected by the calcium phosphate method with 5 μg of pPB(−285/+32)-LUC reporter plasmid along with 1 μg of pSG5-rAR or empty pSG5 and 5 μg of SNURF expression vector (pcDNA-SNURF) or empty expression vector (pcDNA-3.1+) in the presence 25 nM testosterone (T) as depicted. β-Galactosidase expression plasmid, pCMVβ (2 μg), was used as a control for transfection efficiency. (B) CV-1 cells were transfected with 1 μg of pSG5-rAR, 5 μg of pARE₂-TATA-LUC reporter, 2 μg of pCMVβ, and 5 μg of pcDNA-SNURF or empty expression vector in the presence or absence of testosterone (T) as depicted. (C) The experimental conditions were as in panel B, except that 5 μg of pTATA-LUC (devoid of AREs) was used as a reporter. Reporter gene activities are expressed relative to that achieved with pSG5-rAR in the presence of testosterone (100 in panels A and B; 1 in panel C), and the mean ± standard error values of at least three independent experiments are given. (D and E) SNURF activates PR- and GR-dependent transcription. (D) Effect of SNURF on the transcriptional activity of PR. CV-1 cells were transfected with 5 μg of pARE₂-tk-LUC reporter containing two copies of the GRE-PRE-ARE element of the rat TAT gene upstream of the thymidine kinase promoter along with 1 μg of pSG5-hPR1, 5 μg of pcDNA-SNURF or empty expression vector pcDNA-3.1+, and 2 μg of pCMVβ in the presence or absence of 100 nM progesterone (P). (E) CV-1 cells were transfected as for panel D but with 1 μg of pSG5-hGR instead of pSG5-hPR1 in the presence or absence of 100 nM dexamethasone (D). Luciferase (LUC) activities are expressed relative to those achieved with pSG5-hPR1 and pSG5-hGR in the presence of progesterone and dexamethasone, respectively (those values being equal to 100), and the mean ± standard error values of at least three independent experiments are shown. (F) Effect of SNURF overexpression on Sp1 activity. CV-1 cells were transiently transfected with 5 μg of pTATA-LUC, pSp1-TATA-LUC, or pSp1₂-TATA-LUC reporters along with 5 μg of SNURF expression vector (pFLAG-SNURF) or empty expression vector (pFLAG-CMV-2) and also 2 μg of pCMVβ. Transcriptional activities are expressed as relative luciferase (LUC) activity normalized by using the β-galactosidase activity.