Figure 4

Inhibition of the activity of PSA promoter by C/EBPα. A. LNCaP cells were transiently co-transfected with a PSA promoter/enhancer-driven luciferase reporter plasmid and the C/EBPα expression plasmids, C/EBPα-pEGFP and pcDNA3-C/EBPα. After transfection, cells were treated for 36 hrs with 50 nM 5α-dihydrotestosterone (DHT) in RPMI 1640 medium supplemented with charcoal-stripped FCS. Cell extracts were collected with passive lysis solution, luciferase activity measured using pCMV renilla to standardize the transfection efficiency, and the results expressed as the mean relative light units ± standard deviation of 4 separate experiments. Open bars, basal transcription without DHT. Dark bars, transcription with DHT. Open double stars, statistically significant basal transcription in the C/EBPα expressing cells compared with control cells p-value < 0.01. Dark double stars, statistically significant DHT-stimulated transcription in the C/EBPα expressing cells compared with control cells with p-value < 0.01. B. Expression of C/EBPα did not inhibit the activity of PSMA promoter. The PMSA promoter-1(2 kb) and promoter-2 (5 kb) in the pGL-3 luciferase reporter plasmid, kind gifts from Dr. Sidney R. Grimes, were co-transfected with the C/EBPα expression plasmid into ALVA 101 cells. The luciferase assay was conducted as described in Methods.