lamellipodia and actin stress fiber polymerization of HCC cells [15]. However, the underlying mechanism of Pyk2 on regulation of cell transformation and motility of HCC cells is poorly understood. Recently, some of the signaling molecules associated with Pyk2 (Hic-5 and STAT5b) have been reported to promote EMT [16,17]. Therefore, it is valuable to investigate the effect of Pyk2 on regulating these molecular in the process of cell transformation of HCC cells. Hydrogen peroxide inducible clone-5 (Hic-5) is a 55 kDa protein that serves as an adaptor protein in focal adhesion and possesses the ability to translocate to the nucleus, where it acts as a transcription factor [18]. It maintains the general structure of paxillin with 4 N-terminal LD motifs and 4 C-terminal LIM domains [19,20]. The LIM domain of Hic-5 is able to bind with DNA fragments in a zinc-finger-dependent manner in vitro, suggesting its possible role as a transcription factor [21]. Pyk2 can physically interact with Hic-5 and subsequently phosphorylate Hic-5 [22]. It has been reported that activated Pyk2 phosphor- ylates Hic-5 at tyrosine residue 60 resulting in up-regulation of LPA-mediated cell migration and the induction of EMT through a RhoA/ROCK-dependent pathway [17,23,24]. So far the inter- action between Pyk2 and Hic-5 in HCC is still unknown. STAT5b belongs to the Signal Transducers and Activators of Transcription (STAT) family which are activated by cytokines and transcription factors via its dimerization and nuclear translocation. They are involved in a variety of cell processes including cell proliferation, cells survival and differentiation [25,26,27]. STAT5b has been reported to promote EMT in HCC by the transforma- tion of HCC cells into an aggressive phenotype. It has been shown that the activation of STAT5b is closely associated with the X protein of the Hepatitis B virus [16]. In this study, we hypothesize that Pyk2 transformed HCC cells to a fibroblastoid phenotype through regulation of genes associated with EMT. To validate the hypothesis, Hep3B cells were stably overexpressed with full length Pyk2 and MHCC97L cells were stably transfected with PRNK to suppress the Pyk2 activation. The gene expression profiles associated with EMT (Hic-5, STAT5b, E-cadherin, Twist, N-cadherin, fibronectin and Rho GTP-binding proteins) were investigated. Materials and Methods Reagents, plasmids and antibodies Platelet derived growth factor-BB (PDGF-BB) was purchased from Calbiochem (Darmstadt, Germany). Lysophosphatidic acid (LPA) was purchased from Sigma (St. Louis, MO, USA). Plasmids pCDNA3-Pyk2 and pCDNA3-PRNK were gifts from Dr. Joseph Loftus, Mayo Clinic Scottsdale, USA. pCDNA 3.1 (+) vector was purchased from Invitrogen (Carlsbad, CA). Anti-E-cadherin, anti- N-cadherin, anti-Twist and Anti-phosphotyrosine monoclonal antibodies were purchased from Cell Signaling (Danvers, MA, USA). Monoclonal antibodies against cytokeratin (AE1/AE3) was purchased from DAKO (Glostrup, Denmark). Anti-STAT5b and anti-fibronectin antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Anti-Hic-5 and anti-Pyk2 monoclonal antibodies were purchased from BD Transduction Laboratory (San Jose, CA, USA). Alexa fluor 488 goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Molecular Probes (Carlsbad, CA, USA). Cell culture, transfection and stable cell lines Human HCC cell line Hep3B was purchased from the American Type Culture Collection (Manassas, VA, USA) and grown in DMEM medium containing 10% FBS, 2 mM L- Pyk2 on Cell Motility of Hepatocellular Carcinoma glutamine, and 100 units/ml streptomycin (Life technologies, Carlsbad, CA, USA). Human metastatic HCC cell line MHCC97L was a gift from Prof. Z.Y. Tang, Fudan University, Shanghai, China. Stable transfectants MHCC97L-vector and MHCC97L-PRNK has been reported previously. Hep3B cells were stably transfected with full length Pyk2 or PCDNA 3.1 (+) empty vector and maintained in DMEM medium supplemented with 300 mg/ml G418 as described previously [15]. The expressions of Pyk2 in the stable clones were confirmed by Western blotting. One clone with the highest expression of Pyk2 was selected for further study. Scanning electron microscopy (SEM) Cells were seeded in 13-mm glass dishes and maintained in serum-free DMEM medium supplemented with 10% FBS and antibiotics G 418 (0.6 mg/ml for Hep3B cells and 0.2 mg/ml for MHCC97L cells). To study the effects of Rho small GTPases activation on cell transformation, the cells were further treated with PDGF-BB (10 ng/ml) or LPA (1 mg/ml) for 10 minutes before cell fixation. Cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate-HCL buffer, pH 7.4, quenched with 0.1 M sucrose/cacodylate solution and washed in caco- dylate buffer. The samples were then post-fixed with 1% OsO4 in cacodylate buffer. After a cacodylate buffer wash, samples were dehydrated through a graded series of ethanol washes, followed by critical point drying using a Bal-Tec CPD 030 (Bal- Tec AG, Liechtenstein). The samples were sputter-coated with a thin layer of gold (Bal-Tec SCD005 Sputter Coater) and visualized using a Leica Cambridge Stereoscan 440 SEM at an accelerating voltage of 12 kV. Each experiment was repeated for 3 times. Activation assays for Rac1 and RhoA Rac and RhoA activation assays were purchased from Cytoskeleton (Denver, CO, USA). Hep3B transfectants were serum-starved overnight prior to experiment. Cells were treated with PDGF-BB (10 ng/ml) or LPA (1 mg/ml) for 5 minutes and were then lysed with lysis buffer. Rac1 and RhoA pull-down assays were performed according to manufacturer’s protocol. Migration assay Cells were trypsinized, counted and resuspended in serum free DMEM medium. Around 50,000 cells were seeded on the upper side of the migration chamber (BD, San Jose, CA, USA) with the lower chamber supplemented with serum free DMEM medium or with LPA (1 mg/ml). After 36 hrs cells that had penetrated through the chamber were fixed and counted. Immunofluorescence staining The protocol for immunofluorescent staining has been reported previously [15]. Briefly, cells were fixed and stained with anti-E- cadherin, anti-Hic-5 and anti-STAT5b antibodies. The cells were then labeled with alexa fluor 488 goat anti-rabbit IgG or goat anti- mouse IgG and counter-stained with DAPI at 37oC. Immunoprecipitation and Western blotting Immunoprecipitation was performed on whole-cell lysates using antibodies against Hic-5 and phosphotyrosine. The cells were serum starved for 24 hours before stimulation with LPA for 20 minutes. Cell lysates were then incubated with anti-Hic-5 antibody for 4 hours at 4oC. Immunoprecipitates were washed twice in ice- cold lysis buffer. Immunoblotting was carried out as previously reported [15]. PLoS ONE | www.plosone.org 2 April 2011 | Volume 6 | Issue 4 | e18878