Pyk2 on Cell Motility of Hepatocellular Carcinoma Figure 2. Effects of Pyk2 on epithelial genes cytokeratin and E-cadherin. (A) Effects of Pyk2 over-expression on the localization of E-cadherin in Hep3B cells. Positive staining of E-cadherin (green, white arrow) was present in the cytoplasm of the transfectants. (B) Over-expression of Pyk2 down-regulated the expression of cytokeratin and E-cadherin in Hep3B cells as shown by Western Blotting. (C) Forced expression of PRNK in MHCC97L cells restored the expression of E-cadherin (green, white arrow) on the cell membrane and cytoplasm. (D) The expression of E-cadherin and cytokeratin was increased upon suppression of Pyk2 in MHCC97L cells. doi:10.1371/journal.pone.0018878.g002 cytoplasm of Hep3B-Pyk2 cells, indicating the deactivation of E- cadherin. Western blot analysis showed that the expression levels of E-cadherin and cytokeratin were lower in Hep3B-Pyk2 cells compared with that in Hep3B-vector cells (Fig. 2B). In MHCC97L-vector cells with high baseline level of Pyk2, E- cadherin expression was almost absent in the cytoplasm of the cells (Fig. 2C). Transfection of PRNK in MHCC97L cells significantly restored the expression of E-cadherin at cytoplasm and on cell membranes (green, Fig. 2C). Western blot analysis showed that suppression of Pyk2 activation by PRNK restored the expression of E-cadherin and cytokeratin (Fig. 2D) in MHCC97L-PRNK cells. These results confirmed the role of Pyk2 on down-regulation of epithelial genes E-cadherin and cytokeratin. Pyk2 activated Hic-5 and its focal adhesion localization Inmmunofluorescent staining demonstrated that Hic-5 was predominantly present in the cytoplasm and the cell membrane of the Hep3B-vector cells in a dispersed pattern (Fig. 3A). Overex- pression of Pyk2 promoted the membrane localization of Hic-5 in Hep3B-Pyk2 cells. Interestingly, more positive Hic-5 signals were present on the membrane and the peri-nuclear region in the Hep3B-Pyk2 cells (Fig. 3A). To further confirm the up-regulation of phosphorylated Hic-5 by Pyk2 overexpression, immunoprecip- itation was performed to determine the presence of phosphory- lated Hic-5 in cultured cells. The level of phosphorylated Hic-5 was up-regulated in Hep3B-Pyk2 cells, as compared to the Hep3B- vector control (Fig. 3B). In MHCC97L cells, Hic-5 was frequently localized on the membrane region as shown by the strong green positive staining by IF (Fig. 3C). Suppression of Pyk2 activation in MHCC97L-PRNK cells significantly down-regulated the mem- brane localization of Hic-5 (Fig. 3C). Moreover, Western blot result showed that suppression of Pyk2 resulted in the down- regulation of phosphorylated Hic-5 in MHCC97L-PRNK cells (Fig. 3D). These results confirmed the positive correlation between the expression of Pyk2 and the degree of phosphorylation and membrane localization of mesenchymal gene Hic-5 inside the cell. PRNK down-regulated the expression and activation of STAT5b In Hep3B-vector cells with low endogenous expression of Pyk2, green positive immunofluorescent staining of STAT5b was localized in the nuclear region of the cells, indicating its activation. Overexpression of Pyk2 did not significantly enhance the activation of STAT5b because of its intrinsic activation in the vector control (Fig. 4A). Western blot analysis showed that overexpression of Pyk2 in Hep3B did not up-regulate the expression of mesenchymal genes including STAT5b and Twist (Fig. 4B). For MHCC97L-vector cells, STAT5b was predomi- nantly localized at the nucleus. Activated dimerized STAT5b, was present in the nucleus (green; Fig. 4C). Suppression of Pyk2 in MHCC97L-PRNK cells resulted in a loss of nuclear staining of STAT5b. Positive staining of STAT5b was observed in the peri- nuclear region and the cytoplasm, but not in the nucleus (Fig. 4C). Western blot analysis confirmed the down-regulation of STAT5b PLoS ONE | www.plosone.org 4 April 2011 | Volume 6 | Issue 4 | e18878