1. Nuclear Accumulation of Histone Deacetylase 4 (HDAC4) Coincides with the Loss of Androgen Sensitivity in Hormone Refractory Cancer of the Prostate 
K Halkidou, S Cook, H.Y Leung, D.E Neal, C.N Robson 
European Urology 
Volume 45, Issue 3, Pages (March 2004)
DOI: /j.eururo

2. Fig. 1
Western analysis was performed to study the expression of HDAC4 (119kDa) in 3 CaP cell lines. (1) LNCaP cells, AR positive CaP cell line; (2) DU145 and (3) PC3M cells, both AR negative. All cell lines express HDAC4 at comparable levels. No other bands were apparent on the blot, demonstrating antibody specificity. Alpha-tubulin loading control included for each HDAC antibody.
European Urology  , DOI: ( /j.eururo )

3. Fig. 2
LNCaP cell nuclei were stained with the chromatin dye DAPI (blue) while endogenous HDAC4 was detected by indirect immunofluorescence and visualised as red signal. Cells were divided into four groups and cultured for 72hours in full medium containing androgens (FM) (A); in steroid deprived medium (SDM) (B); in SDM supplemented with 10nM R1881 for a 3-day period (C); or in bicalutamide (10μM) containing FM (D). HDAC4 normally resides in both cellular components (A), while, in the absence of androgens, HDAC4 exclusively localises to the cytoplasm (B), with no red/blue signal overlap. Cytoplasmic HDAC4 expression was also observed upon anti-androgen treatment (D), suggesting AR is directly implicated in the above response. Interestingly, HDAC4 responds to androgen treatment by nuclear translocation ((C) complete overlap of the red signal with the blue counterstain). = 10μm. (E) Fluorescent signal quantification was performed in order to further support the nuclear cytoplasmic translocation of endogenous HDAC4 in LNCaP cells when in SDM, compared to FM. Three individual experiments were performed and at least 10 cells per case were measured using the “area” tool of the Leica software. The DAPI (blue) counterstain was used as a guide to plot the nuclear area in each case. The difference of the means was calculated and a p-value of was found between SDM and FM. Therefore, there is a statistically significant downregulation of HDAC4 expression in the nucleus upon SDM treatment. Similarly, the cytoplasmic signal increases proportionally when in SDM (data not shown).
European Urology  , DOI: ( /j.eururo )

4. Fig. 3
CWR22 mouse CaP xenograft model. While androgen deprivation led to cytoplasmic expression of HDAC4 (castrated mice group, data not shown), testosterone supplementation resulted in increasing nuclear translocation upon increasing treatment periods (T1–T4). From mainly cytoplasmic in the untreated control (U1) and the first two weeks of testosterone treatment (T1 and T2, respectively), HDAC4 gradually became nuclear after 3 and 4 weeks of exposure to androgens (T3 and T4, respectively). Scale bar = 30μm.
European Urology  , DOI: ( /j.eururo )

5. Fig. 4
Immunohistochemistry staining of HDAC4 (brown signal) in human prostate tissue and control sections form breast and ileum. Haematoxylin counterstain of the nucleus (blue signal). (A) HDAC4 is predominantly expressed in the cytoplasm of benign glands. (B) In androgen responsive primary cancer of the prostate HDAC4 resides mainly in the cytoplasm, however, nuclear signal is detected in a small subpopulation of malignant cells (arrows). (C) The expression profile is altered in hormone refractory cancer, where the malignant prostate glands show a primarily nuclear signal of HDAC4 in all 10 cases studied. No detectable cytoplasmic signal was observed in any case of HR CaP. Note the large, irregularly shaped tumour glands in and the infiltration of malignant cells into the surrounding stroma (asterisks). (D) Using the ×100 oil immersion lens, HDAC4 signal is shown to be diffusely nucleoplasmic without nuclear aggregate formation or sequestration in subnuclear compartments. (E and F) To assess the specificity of HDAC4 antibody, positive controls were carried out in tissues shown to strongly express HDAC4 in the cytoplasmic compartment, such as smooth muscle cells. Specifically, normal breast and ileum tissue was stained for HDAC4. As shown in (E), smooth muscle cell bundles (arrows) surrounding a vessel (asterisk) within normal breast tissue, show strong cytoplasmic expression of HDAC4. Similarly, the smooth muscle cell layer of the ileum (asterisks in (F)) strongly expresses HDAC4 in the cytoplasm, as previously reported. Additionally, smooth muscle cell bundles within the stromal compartment of BPH sections, displayed strong cytoplasmic signal and served as internal positive controls (data not shown). Scale bar = 30μm.
European Urology  , DOI: ( /j.eururo )