Figure 1

CEBPD is an androgen-responsive gene. (a) The level of CEBPD is associated with the abundance of the AR. RT-PCR analysis and a western blot were conducted to detect the expression of CEBPD and AR, as indicated in LNCaP and PC-3 cells in DMEM supplemented with FBS (without charcoal treatment). (b) DHT stimulates the expression of CEBPD in LNCaP cells but not in PC-3 cells. RT-PCR analysis and western blot were conducted to detect the expression of CEBPD and AR at the indicated time. The LNCaP and PC-3 cells were cultured in DMEM supplemented with charcoal-treated FBS. (c) DHT activates the activity of a CEBPD reporter in LNCaP cells. LNCaP cells were transfected with the reporter pGL2-Basic vector carrying fragments of the CEBPD promoter (−1720 to +18 bp, −1000 to +18 bp or −360 to +18 bp). After 16 h of transfection, the transfectants were treated with 10 nM DHT for 6 h and the cell lysates were harvested for luciferase assay. Columns, the average of three independent experiments in duplicate measurements; bars, mean±S.D. (**P<0.01, NS: no significance; Student’s t-test). (d) The AR binds to the CEBPD promoter in vivo. A ChIP assay was conducted with the chromatin of LNCaP cells after 4 h of treatment with 10 nM DHT. The chromatin of experimental cells was separately immunoprecipitated with control or anti-AR antibodies. The precipitated DNA was then purified and amplified by PCR with the −802 and −567 primers targeting the CEBPD gene locus as indicated. Numbers below the images are the levels normalized to GAPDH (mRNA) or α-tubulin (protein)