Somatic AR mutation and amplification in E006AA cells.

<p>(A) Cytogenetic analysis of AR status in normal male control and E006AA cells as determined by Fluorescence in Situ Hybridization (FISH) using probes specific for the AR Gene (Xq12, red colored) and the X centromere (green colored). (B) Sequencing results of overlapping primer PCR amplification of E006AA mRNA and cDNA identified a missense mutation (Ser599Gly), which corresponds to the “X” position of the conserved AXRND motif in the DBD D-box (as denoted by black arrowhead at position 597 of the reference human AR NM_000044), as found in human AR, GR, PR, and MR. (C) Quantitative PCR analysis of AR mRNA transcript levels in LNCaP compared to E006AA cells. (D) Western blot analysis of AR protein expression in E006AA cells infected with lentiviral non-silencing shRNA, wild-type AR (Lenti-AR), or anti-AR shRNA (AR shRNA) constructs compared to parental E006AA, LNCaP, PC-3 and S006AA stromal cells. Protein lysates were loaded on a per cell basis as follows: LNCaP (20,000), all others (200,000) cells per lane.