Figure 3. The AR and HDAC6 inhibitory activity of Zeta55 in cells. The agonistic effects (A) and antagonist effects (B) of Zeta55 on AR by reporter gene assay. The VCaP cells were transfected with MMTV-LUC for 24 hours, and then treated with Zeta55 or MDV3100 in the absence of DHT for agonistic mode or with DHT for antagonist mode. Zeta55 decreases RNA expression level of PSA (C) and TMPRSS2 (D) in VCaP cells. Zeta55 also decreases and protein expression level of PSA (E) and TMPRSS2 (F) in VCaP cells. The VCaP cells were treated with Zeta55 or MDV3100 together with 10 nM DHT for 24 hours. RNA expression level was analyzed by qRT-PCR and protein expression level was analyzed by Western blot. Zeta55 increases acetylation of α-tubulin (G) and decreases AR (H) in VCaP cells. VCaP cells were treated with Zeta55, MDV3100 or SAHA for 24 hours before Western blot analysis. (I) Representative immunofluorescence microscopic images of VCaP cells for checking the subcellular localization of AR. VCaP cells were cultured with 10% FBS and treated with vehicle (control group),10 μM MDV3100 or Zeta55 for 24 hours, and then stained with DAPI (4’,6-diamidine-2’-phenylindole) and AR.