Figure 3. Cyclin D1b interacts with AR on chromatin and enhances AR occupancy at SNAI2 regulatory loci.

(A) Androgen-depleted LNCaP cells were stimulated with 1 nM DHT for the indicated time points and relative expression of SNAI2 and KLK3 transcript levels determined. (B) Androgen proficient LNCaP lysates expressing a 3× flag cyclin D1b construct were fractionated into soluble and chromatin-tethered lysates and subjected to immunoprecipitation of AR or Flag. 10% input and IgG served as positive and negative controls, respectively, while GAPDH and histone H4 served as soluble and chromatin-tethered specific controls, respectfully. (C) LNCaP vector and cyclin D1b cells were androgen depleted for 72 hours and then stimulated with either DHT (10 nM) or EtOH (0.1%) for 3 hours. Samples were harvested for ChIP analysis, and percentage (input) occupancy of AR (top panel), cyclin D1b (middle), and acetylated histone H4 (bottom panel) are reported for the SNAI2 and the KLK3 loci (D). Error bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001