CHIP Assay
SimpleChIPH Enzymatic Chromatin IP Kit (Magnetic Beads) and 6-Tube Magnetic Separation Rack were purchased from cell signaling and used for performing CHIP assay according to the manufacturer’s instruction. Briefly, PC3 cells treated with 400 nM TSA and 5 mM SAHA or DMSO control for 16 hours and 37% formaldehyde was added (Final formaldehyde concentration was 1%) to crosslink proteins to DNA and mixed, and then incubated for 10 minutes at room temperature. Glycine was added into dishes and the cells were incubated for 5 minutes at room temperature. After washing with ice-cold PBS, the nuclear extracts from the cells wee prepared. For cross-linked chromatin preparation, the nuclear suspension was added to Micrococcal Nuclease to digest DNA to length of approximately 150?00 bp and Micrococcal Nuclease was inactivated by EDTA, and then sonicated with several pulses to break nuclear membrane. After
Western Blot Analysis
Total cell lysates were prepared by lysing the cells in RIPA buffer. Protein concentrations were measured using protein assay reagents (Pierce, Rockford, IL). Proteins with equal amount were subjected to SDS-PAGE to separate and electrophoretically transferred to nitrocellulose membranes. After blocking with 3% milk, the protein was detected by incubating with respective primary antibodies followed by incubation with respective secondary antibodies as described previously [26].
Real-time RT-PCR
The total RNA was isolated using RNeasy mini kit (Qiagen, Valencia, CA). The residual DNA was removed using an RNase-
Figure 2. TSA and SAHA increased the expression of EMT-related transcription factors and mesenchymal markers in PCa cells at different doses. Total RNA was isolated from PC3 cells treated with 200 nM and 400 nM TSA (A) or 2.5 mM and 5 mM SAHA (B) for 24 h. The results from real time RT-PCR showed that the relative mRNA expression of ZEB1, ZEB2, Slug, Twist, N-Cadherin, vimentin and MMP2 was increased following treatment compared to DMSO control (the value of control was designed as 1, * p,0.05, ** p,0.01).purification, DNA fragment size was determined by electrophoresis on a 1% agarose gel and results showed that ranges of DNA size was approximately 150?00 bp length. For chromatin immunoprecipitation, 15 mg of chromatin DNA in CHIP buffer (for each immunoprecipitation) was incubated with the immunoprecipitating antibody: Ac-H3, HDAC1, HDAC2, HDAC3, HDAC4, HDAC6, and the negative control (Normal Rabbit IgG) at 4uC with rotation. After incubation overnight, 30 ml of ChIP Grade Protein G Magnetic Beads were added and incubated for 2 h at 4uC with rotation Chromatin was eluted from Antibody/Protein G Beads and cross-links was reversed by incubating with Proteinase K 2 hours at 65uC. DNA was purified by using Spin Columns. Quantification of DNA was performed by PCR using specific primers for gene promoter of vimentin, ZEB2, Slug and MMP2. Primer sequences for these gene promoters are shown in the table 1. The relative amount of promoter DNA was normalized to the input. IgG was used as reference control and calculated as unit value of 1.0.
Results HDACIs Induced EMT Phenotype in Prostate Cancer Cells
To investigate the effects of HDACIs on cell morphology, we treated PC3 cells with TSA or SAHA, and we found that PC3 cells treated with TSA or SAHA displayed elongated/irregular fibroblastoid morphology. In contrast, PC3 cells treated with DMSO exhibited a cobblestone appearance, typical morphology of epithelial cells (Fig. 1A and B). DU145 and ARCaPE cells treated with TSA or SAHA also displayed elongated/irregular fibroblastoid morphology (Data not shown). These results suggest that treatment of prostate cancer cells with HDACIs leads to the induction of EMT phenotype. To further confirm whether HDACIs could really induce EMT in PC3 cells, we determined the expression of vimentin and ZEB1 using immuno-fluorescence staining. TSA and SAHA induced EMT phenotype was consistent with increased expression of vimentin and ZEB1 (Fig. 1C, upper and middle panel). In addition, F-actin reorganization and a diffuse pattern were also observed in PC3 cells treated with TSA and SAHA (Fig. 1C, lower panel), which were correlated with EMT phenotype.
Immuno-fluorescence Microscopy
Immuno-fluorescence staining was performed as described previously by our laboratory [18]. Briefly, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature (RT). After washing 3 times with PBS, the cells were permeabilized in 0.5% Triton x-100 in PBS for 10 minutes, and then blocked with 10% goat serum. The cells were incubated for 40 minutes with antibodies against vimentin (pre-diluted) and ZEB1 (1:50) in 5% goat serum, washed and then incubated for another 40 minutes with Alex Fluro 594 conjugated secondary antibody (1:250) at room temperature. For F-actin staining, the cells were fixed with 4% paraformaldehyde for 15 minutes and permeabilized in 0.5% Triton x-100 in PBS for 10 minutes, and then incubated with 0.33 mM of Alexa Fluor 594 phalloidin for 40 minutes at 4uC. After washing, the slides were mounted with mounting medium containing anti-fade reagent and DAPI. The cells were viewed under fluorescence microscope. The images were captured and analyzed by using Advanced Sport software (Diagnostic Instruments, Sterling Heights, MI).
