Statistics and data analyses All data were expressed as mean 6 SD. Difference between groups were calculated by one-way ANOVA analysis. A p,0.05 was considered statistically significant. Unless stated, all experi- ments were repeated three times. Calculations were performed by using the SPSS computer software version 12 (SPSS, Chicago, IL). Results Pyk2 promoted formation of membrane ruffles Forced overexpression of Pyk2 in the non-metastatic HCC cell line Hep3B (Hep3B-Pyk2 cells) enhanced the formation of lamellipodia and membrane ruffles upon stimulation by PDGF- BB or LPA compared to control Hep3B-vector cells (Fig. 1A). Suppression of Pyk2, by overexpression of PRNK domain, in the metastatic MHCC97L cells (MHCC97L-PRNK cells) reduced the formation of membrane ruffles, as compared to the MHCC97L- vector control (Fig. 1B) in presence of PDGF-BB or LPA. Migration assay demonstrated that Hep3B-Pyk2 exhibited a significant increase of LPA-induced migration than Hep3B-vector (p,0.05, Fig. 1C). Suppression of Pyk2 activation by PRNK Pyk2 on Cell Motility of Hepatocellular Carcinoma significantly down-regulated the cell migration in MHCC97L- PRNK cells as compared to the vector control (p,0.05, Fig. 1C). To elucidate the contributing mechanisms to the increase of membrane ruffles formation in Hep3B-Pyk2 cells, Rac1 and RhoA pull-down assays were performed to study the effects of Pyk2 on LPA-induced activation of Rac1 and RhoA. Significant up- regulation of activated forms and baseline levels of Rac1 and RhoA were found in Hep3B-Pyk2 cells compared to Hep3B- vector cells (Fig. 1D). For MHCC97L cells, no activated Rac1 and RhoA was detected in their cell lysates due to low baseline levels of Rac1 and RhoA (data not shown). Pyk2 down-regulated the expression of E-cadherin and cytokeratin The effects of overexpression of Pyk2 on the regulation of epithelial genes E-cadherin and cytokeratin were investigated. Result from immunofluorescent staining showed that Hep3B-Pyk2 cells had lower level of E-cadherin expression and localization on the cell membrane as compared to the Hep3B-vector cells (Fig. 2A). Weak positive staining (green) was present in the Figure 1. Pyk2 promoted cell motility of HCC cells. (A) Overexpression of Pyk2 in Hep3B cells enhanced the formation of membrane ruffles while (B) suppression of Pyk2 in MHCC97L cells reduced the formation of membrane ruffles under the stimulation of PDGF and LPA. White arrows indicate the presence of membrane ruffles. (C) Effects of Pyk2 on cell migration of Hep3B and MHCC97L cells in presence of LPA. *, P,0.05. (D) The effect of over-expression of Pyk2 on the activation of Rac1 and RhoA in Hep3B cells by Rac1 and RhoA pull-down assays. Total Rac1 and RhoA were used as loading controls. doi:10.1371/journal.pone.0018878.g001 PLoS ONE | www.plosone.org 3 April 2011 | Volume 6 | Issue 4 | e18878