Figure 3

Expression of C/EBPα inhibits PSA expression through inhibition of the PSA promoter. A. Increasing expression of C/EBPα reduced protein levels of PSA in transduced LNCaP cells. The Clone 2 cells at passage 3 and the corresponding control cells were treated with 500 nM trichostatin A (TSA) for 48 hours. Whole cell protein extracts were again subjected to SDS-PAGE and western blot analysis performed. The expression of PSA and C/EBPα were expressed relative to β-tubulin by densitometry of the western blots. The results in the right panels are the means ± standard deviation of three independent experiments. For PSA expression a single star indicates a significant difference with a P < 0.05 (unpaired t-test) comparing C/EBPα to C/EBPα plus TSA. For C/EBPα expression double stars indicate a significant difference with a P < 0.01 (unpaired t-test). B. LNCaP cells were transiently transfected either with pEGFP-N2, the transfection control, or C/EBPα-pEGFP. After 48 hours EGFP positive cells were collected by cell sorting and RNA extracted from the EGFP positive cells. The PSA mRNA was amplified with RT-PCR as detailed in the Methods. In the left panel is shown the electrophoresis pattern of the PCR products from the cells transfected with pGFP-N2 (lane 1) and C/EBPα-pEGFP (lane 2). A densitometric scan of the electrophoretic pattern corrected for levels of GAPDH shows the relative amounts of PSA mRNA in the control versus C/EBPα transfected cells (right panel) with the columns in the right panel representing the means of two individual PCR reactions.