(A): RT-PCR and semi-quantitative expression levels of cyclin dependent kinase inhibitors p27 and p21 in DU145, DU145-Id4 and PrEC (normal prostate epithelial) cells. (B) Semi-quantitative analysis of RT-PCR results shown in (A). The intensity of each p27 and p21 band was normalized to constitutively expressed beta actin. The RT-PCR data and semi-quantitative analysis (expressed as mean ± SEM) is representative of 3 different RT and corresponding PCR reactions (***: P < 0.001, *: P < 0.05 as DU145). The following primers were used: p27 (CDKN1A): Forward 5'-TCA AAC GTG CGA GTG TCT AA and Reverse 5'-ACG TTT GAC GTC TTC TGA GG, p21: Forward 5'-CGA CTG TGA TGC GCT AAT G and Reverse 5'-TTA GGG CTT CCT CTT GGA GA, β-actin: Forward-5' AGA AAA TCT GGC ACC ACA CC, Reverse-5' GGG GTG TTG AAG GTC TCA AA. (C): Real time analysis of Id4, p53 and E-cadherin gene expression in DU145, DU145-CMV and DU145-Id4 cell lines. The data performed in triplicate is mean ± SEM from cells at passages 18, 20 and 23. The real time data is normalized to the constitutively expressed gene beta-actin. The relative expression levels were calculated by the Δ Ct method as described in materials and methods section. The RT-PCR data is representative of 3 different RT and corresponding PCR reactions (***: P < 0.001, as compared to DU145).