Reciprocal regulation between MID1 and AR. (A) MID1 protein levels in response to androgen and anti-androgen treatment. Western blot analysis (upper part) detecting MID1, AR and GAPDH after treatment with the synthetic androgen R1881 or the androgen antagonist bicalutamide at indicated time points. Lower part: bar-graph of densitometric analysis of respective Western blots from 3 experiments. (B) MID1 mRNA levels in response to androgen and anti-androgen treatment. Real-Time PCR analysis of MID1 relative to GAPDH mRNA levels in DuCaP cells after treatment with R1881 or bicalutamide. (C) The MID1 gene comprises androgen response elements (AREs) inside its promoter and in intronic regions. Graphical demonstration of AR binding sites determined by Chromatin Immunoprecipitation (ChIP) coupled with sequencing in the AR positive DuCaP cell line. Differential peaks between control and androgen treated panels indicate AR binding to these regions. Arrows show AR binding sites, circles show binding sites selected for functional validation. (D) Confirmation of selected AR binding sites in an independent ChIP sample set. Selected AR binding sites (intronic and 105 kb distal promoter sites) were amplified with specific primers and the enrichment of AR bound fragments was determined by Real-Time PCR in ChIP samples in response to androgen (R1881) or vehicle treatment. (E) Functionality of AREs in the MID1 gene. AR-dependent luciferase reporter assays in AR-positive DuCaP cells were performed to assess the function of the 105 kb distal promoter and the intronic AR binding sites. Reporter vectors carrying either binding site DNA fragments or the binding site sequences with mutations introduced into the predicted AREs were transfected into DuCaP cells and cells were treated with R1881 or vehicle control for 24 h. (n = 3).