Figure 3

The SUZ12/EZH2-associated histone K27 tri-methylation attenuates DHT-induced CEBPD transcription in PrCa cells. (a) The expression of CEBPD and AR in LNCaP 104-S and 104-R1 cells. RT-PCR and western blot assays were conducted to detect the expression of CEBPD and AR. The LNCaP 104-S (androgen sensitive) and 104-R1 (androgen insensitive) cells were cultured in DMEM supplemented with FBS without charcoal treatment. (b) The endogenous EZH2, E2F1, SUZ12, DNMT1, DNMT3a, DNMT3b and H3K27me3 expression in LNCaP 104-S and 104-R1 cells. Western blot assays were conducted to detect the expression of protein levels. The LNCaP 104-S (androgen sensitive) and 104-R1 (androgen insensitive) cells were cultured in DMEM supplemented with charcoal-treated FBS. (c) The DNA methyltransferase inhibitor 5-AzadC does not reverse the transcription of CEBPD in LNCaP 104-R1 cells. RT-PCR and western blot assays were conducted to detect the expression of CEBPD in response to 5-AzadC in LNCaP 104-S or 104-R1 cells. Two independent experiments were conducted and showed a similar pattern. (d) DHT-stimulated CEBPD transcription was enhanced in cells that were treated with the histone methyltransferase inhibitor DZNep. LNCaP 104S and 104-R1 cells incubated with 1 μM DZNep. After 18 h of treatment, cells were treated with 10 nM DHT and the cell lysates were harvested at the indicated time to perform RT-PCR with specific primers. Three independent experiments were conducted and statistically plotted. Columns, the average of three independent measurements; bars, mean±S.D. (**P<0.01; Student’s t-test). Numbers below the images are the levels normalized to GAPDH (mRNA) or α-tubulin (protein)