Diaminobenzidine (DAB) was used as the chromogen and counterstaining was done with Mayers hematoxylin. Following three sellckchem 5 min washes, cellscoverslips were mounted on slides with coverslips.For TUNEL assays, fixed cells were incubated with an equilibrium buffer for 5min using the in situ apoptosis detection kit, Fluorescein (Apoptag; Roche, BMS), and then treated in reaction buffer with 10 units of terminal deoxynucleotidyl transferase and 1 unit of deoxyuridine triphosphate digoxigenin at 37��C for 1 hour. The reaction was terminated by adding stop/wash buffer and then washed twice with Tris buffer. Anti-digoxigenin-FITC was added and reacted at 37��C for 30min. After washing with distilled water, nuclei were counterstained with Hoechst 33258 (Sigma Chemical, St Louis, MO), and apoptosis in the cells was observed under a fluorescent microscope.

Cells with green fluorescent (FITC) colored nuclei were considered apoptotic. For quantifying apoptotic cells, apoptotic and total cells were counted in 5 random fields scoring between 300 and 500 cells, and the numbers of apoptotic cells were expressed as percentages of the total cell population. Immunocytochemical staining slides and TUNEL staining slides were observed with microscope (TE-300, Nikon, Japan).2.6. Statistics AnalysisResults were analyzed using a two-tailed Student’s t-test to assess statistical significance. Values of P < 0.05 were considered statistically significant.3. Results3.1.

Androgen Receptor siRNA Transfection Efficiency To explore the feasibility of siRNA transfection efficiency in knocking down AR expression in prostate cancer cells that harbor the AR gene, fluorescent oligo staining with BLOCK-iT was used and the green staining of more than 95% of cells was confirmed in siRNA transfected cells under fluorescence microscopy (Figure 1(a)). We designed and synthesized three siRNAs, AR-1, AR-2, and AR-3, against human AR gene. After 48 hours transfection with three sequence-specific siRNAs, one relatively potent siRNA, AR-1, was identified in knocking down AR expression compared with others by checking the mRNA with RT-PCR. This knocking down effect was sequence-specific event because a negative control siRNA with a scrambled sequence had no effect on AR expression level (Figure 1(b)).Figure 1Effective silencing of the androgen receptor (AR) gene expression in ls-LNCap cells after small interfereing RNA (siRNA) treatment.

(a) Transfection efficiency shown by fluorescence microscopy; (b) RT-PCR band of AR from si-ls-LNCap cells after siRNA. …3.2. Immunocytochemistry of MarkersAmong the four experimental cell lines (es-LNCaP, ls-LNCaP, scr-ls-LNCaP, and si-ls-LNCaP), we confirmed the expression level of five prostate cancer related proteins (AR, HSP27, CLU, GRP78, and c-FLIP) with immunocytochemical Cilengitide staining (Figure 2).