Restored AR expression in LNCaPRANKLcells grown in 3-D suspension increases cell adhesion to ColI. (A) IHC analysis indicates that the suppressed AR expression in LNCaPRANKL cells could be restored, when the cells were grown as a xenograft tumor or in 3-D suspension culture. Arrows denote AR nuclear localization. (B) Restored AR in LNCaPRANKL cells grown in 3-D suspension was confirmed with western blotting. While AR expression is high in LNCaPNeo cell grown in 2-D monolayer or 3-D suspension, LNCaPRANKL cells express AR protein only under 3-D suspension conditions. (C) The restored AR is biologically functional since it could promote PSA promoter activity as detected by a PSA promoter-luciferase reporter assay, which showed 4.3-fold increased activity in 3-D suspension culture compared to the LNCaPRANKL cells in 2-D monolayer. Each black bar represents relative light unit (RLU) as a fold difference of 3-D suspension/2-D monolayer for each cell line. (D) The function of the restored AR protein in regulating cell adhesion was indicated by treating LNCaPRANKL cells with the synthetic androgen R1881 (10 nM, 48 hours), which resulted in significantly enhanced cell adhesion to ColI. On the other hand, the antagonistic effect of the anti-androgen casodex (20 nM) was seen mainly under 3-D suspension culture conditions. Each value is the mean ± SD of 2 independent experiments done in triplicate.