Figure 2.

AR reprograms mTOR interaction with the genome of PCa cells. (A) Motif discovery analysis of mTOR ChIP-seq peaks identified from R1881-treated LNCaP cells revealed the ARE as the major motif enriched in response to androgen stimulation. (B) Overlap between AR and mTOR DNA-binding sites following R1881 or vehicle treatment. (C) ChIP-qPCR of mTOR and AR at the same binding sites in LNCaP cells following a 48 h of androgen treatment. (D) ChIP-qPCR analyses of AR and mTOR binding in LNCaP cells transfected with siControl (siC) or siAR and treated with vehicle or R1881 for 48 h. ChIP-qPCRs were performed for androgen-sensitive (left) and androgen-insensitive (right) mTOR-binding sites. (E) Luciferase reporter assay under the control of 2xAREs in LNCaP cells following a 24 h of treatment with R1881, rapamycin, torin 1, or vehicles, as indicated. Results are shown as the average of three independent experiments performed in triplicate. (F) ChIP–reChIP analysis shows corecruitment of AR and mTOR to the same genomic regions following androgen treatment. The significant enrichments at SLC26A3 and mTOR genes with IgG as a second antibody reflect the enrichment from the first ChIP, done with either AR or mTOR antibodies. Results are shown as the average of two independent experiments. (G) Co-IP of AR and mTOR in the absence or presence of androgens. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.