AR restoration was due to the suppression of AP-4 transcription factor upon 3-D suspension culture. (A) 3-D suspension culture provided a suppressing condition for the differential AP-4 expression observed in RANKL-overexpressing cells in 2-D monolayer culture. LNCaPRANKL cells transfected with AP-4 siRNA could further reduce AP-4 expression as shown with qRT-PCR analysis. (B) AR and AP-4 appeared to have an inverse relationship of expression as detected with qRT-PCR analysis. (C) Western blotting analysis was conducted to demonstrate AR restoration upon AP-4 suppression by specific siRNA knockdown. (D) The effect of AP-4 appeared to modulate AR transcription, as determined with transient AR promoter reporter luciferase assay. For each condition, RLU was shown as the fold difference of AP-4 siRNA/SC siRNA transfection. (E) Effect of AP-4 on AR function was assessed by PSA-promoter reporter luciferase assay. In this experiment, AP-4 was first knocked down in LNCaPRANKL cells. AR responsiveness to androgen R1881 was then measured by PSA promoter reporter luciferase assay.