AR in E006AA cells undergoes nuclear translocation, binds DNA but is not transcriptionally activity due to the S599G loss-of-function mutation.

<p>(A) Western blot analysis of nuclear and cytoplasmic AR expression in LNCaP C4-2B, PC-3, and E006AA cells, treated with or without androgen. E006AA fractions were blotted for Vinculin and HDAC to confirm suitable cytoplasmic and nuclear separation, respectively. (B) PCR amplification of a putative ARE in the PSA promoter region from anti-AR immunoprecipitated chromatin from LNCaP C4-2B and E006AA cells, treated with or without androgen. Chromatin immunoprecipitated with an IgG isotype served as a negative control. (C) Quantitative PCR analysis of anti-AR antibody immunoprecipitated chromatin from LNCaP C4-2B and E006AA cells, treated with or without androgen: (Ci) PSA promoter ARE amplification in C4-2B cells; (Cii) PSA enhancer ARE III amplification in C4-2B cells; (Ciii) PSA promoter ARE amplification in E006AA cells; (Civ) PSA enhancer ARE III amplification in E006AA cells. The results are presented as percentage of input. (*, p-value <0.05, Student's T-test). (D) Analysis of AR transcriptional activity in E006AA cells compared to LNCaP C4-2B, PC-3, E006AA Lenti-AR, E006AA Lenti-non-silencing-shRNA, E006AA Lenti-AR-shRNA cells, treated with or without androgen. (E) Introduction of mutant S599G AR into androgen-responsive, PSA producing LNCaP C4-2B cells, and its effect on AR-driven PSA-Luciferase activity in C4-2B cells, with or without androgen (*, p-value <0.05, Student's T-test). All experiments in this section were performed with cells grown in 10% csFBS supplemented media, +/−24 hr stimulation with 1.0 nM R1881.