The lower side of the chamber was filled with DMEM supplemented ref 3 with 10% fetal bovine serum and allowed to incubate at 37��C for 36h. Cells penetrated through the chamber were stained with 0.1% crystal violet and counted. Extraction of nuclear and cytosolic protein Nuclear and cytosolic proteins were extracted by ProteoExtract? subcellular proteome extraction kit (Calbiochem, EMD Biosciences Inc., Merck KGaA, Darmstadt, Germany) according to manufacturer’s protocol. Immunoblotting For immunoblotting, fractions were denatured in sample buffer, and resolved in 8�C15% SDS-polyacrylamide gel. The proteins were transferred to PVDF membrane (Millipore, Billerica, MA, USA), and immunoblot analysis was performed using antibodies indicated in figure legends.

Immune complexes on PVDF were detected by enzyme-linked chemiluminescence (Amersham Biosciences Corp., Piscataway, NJ, USA). Animal studies An orthotopic liver tumour model in nude mice with higher potential of local (intrahepatic) and distant (lung) metastases was established (Lee et al, 2005). Briefly, approximately 1 �� 107 MHCC97H cells in 0.2ml culture medium were injected s.c. into the right flank of the mice, which were then observed daily for signs of tumour development. Once the subcutaneous tumour reached 1�C1.5cm in diameter, it was removed and cut into about 1�C2mm cubes which were implanted into the left liver lobe of another group of nude mice. Liver tumour and lung tissues were harvested for immunostaining of Pyk2 at 5 weeks after tumour implantation. The liver tumour and lung metastases were confirmed by H&E staining.

The protocol for the in vivo animal study has been approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of the University of Hong Kong with meeting the standards required by the UKCCCR guidelines. The project number is CULATR-1133-05. Statistical analysis The ��2 test was used to compare discrete variables. Mann�CWhitney U-test was used for statistical comparison for continuous variables. Kaplan�CMeier method with log�Crank test was used for survival analysis. P<0.05 was considered statistically significant. Calculation was made using SPSS computer software version 12 (SPSS, Chicago, IL, USA).

RESULTS Protein and gene expression of Pyk2 and FAK in HCC patients: distinct expression pattern of Pyk2 and FAK Different from the previous studies (Stanzione et al, 2001; Lipinski AV-951 et al, 2005), positive Pyk2 signals were found both in the cytoplasm and nuclear of tumour cells of HCC patients (Figure 1A). Focal adhesion kinase mainly expressed in the cytoplasm of liver cancer cells (Figure 1B). According to the immunostaining results, there were 29 (59.2%) patients with higher expression levels of Pyk2 and 28 (57%) patients with higher expression of FAK (Figure 2). The protein expression by Western blot was consistent with the immunostaining results (Figure 3).