Figure 2

Microarray analysis of DDC-regulated genes. Experimental outline of microarray studies to identify DDC downstream targets using LNCaP-DDC stable and control LNCaP-pDEST cells. In the presence of Dox (grey triangle, + Dox) the tetracycline Tet repressor (tetR) is released from the TetO2 sequence in the promoter of the lentiviral construct containing the DDC gene. The dissociation of the tetR allows induction of transcription for the gene of interest. LNCaP-DDC and LNCaP-pDEST (vector control) cell lines were plated in medium containing 5% charcoal-stripped serum and incubated overnight. The next day, cells were treated for 48 hours under mock-induced (-Dox) and Dox-induced (+Dox) conditions, 24 h of which was in the presence or absence of 0.1 nM R1881. Total RNA samples were isolated from each condition, labeled and hybridized on the microarrays. The relative mRNA abundance of each gene was calculated as a ratio between hormone-treated (+R1881) and hormone-untreated (- R1881) samples. The comparison of the expression data obtained from LNCaP-DDC+Dox stable cells (left) with the expression data from the three LNCaP control cells (right: LNCaP-DDC-Dox, LNCaP-pDEST- Dox, and LNCaP-pDEST+Dox) yielded the identification of genes that are androgen- and DDC-regulated.