About 0.5��g total RNA from each sample was used to perform reverse transcription (RT) reaction Tofacitinib alopecia using TaqMan Reverse Transcription Reagents (Man et al, 2003) (Applied Biosystems Limited, Foster City, CA, USA). RT product (1��l) was used to perform real-time quantitative RT�CPCR using TaqMan Core Reagent Kit (Applied Biosystems) by the ABI PRISM 7700 Sequence Detection System (Applied Biosystems) (Man et al, 2003). The probes and primers of Pyk2, FAK, ezrin and fibronectin were commercially available from Applied Biosystems limited. The TaqMan Ribosomal RNA Control Reagent (18S RNA probe and primer pair; Applied Biosystems) was used for internal control in the same PCR plate well to normalise the target gene amplification copies. All samples were performed in triplicates.

The relative gene expression levels were calculated as the ratio of the expression of tumour or non-tumour tissues to normal liver from healthy living donors. Functional studies Reagents and plasmids Plasmids PCDNA3-Pyk2 and PCDNA3-PRNK (C-terminal of Pyk2) were gifts from Dr Joseph Loftus, Mayo Clinic Scottsdale, Scottsdale, USA). PCDNA 3.1 (+) vector was purchased from Invitrogen. Monoclonal antibody against Pyk2 (clone 11) was purchased from BD Transduction Laboratories. Polyclonal antibodies against Phospho-Pyk2 (Tyr402) was purchased from Cell Signalling (Beverly, MA, USA). PLC cells were purchased from ATCC and was grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS, 2mM L-glutamine, 100unitsml?1 streptomycin (Life Technologies, Carlsbad, CA, USA).

Metastatic HCC cells MHCC97L was a gift from Dr Tang, Shanghai Fudan University (Lee et al, 2005). Cell culture, transfection and stable cell lines Cells were seeded in six-well plates to 80% confluence. The cells were transfected with 1��gwell?1 PCDNA 3.1, PCDNA-Pyk2, PCDNA-PRNK plasmids using Fugene 6 (Roche, Basel, Switzerland). After transfection overnight, cells were changed to normal medium and allowed to recover overnight. The cells were trypsinised and seeded into new 10cm culture dish and allowed to recover for 2 days. Cells were then grown in DMEM containing G418 at 0.6mgml?1 until all the non-transfected cells were dead (21 days). Resistant clones were spread in 24-well plates using cloning cylinders and maintained in 0.3mgml?1 G 418 for further study. Proliferation assay Cells were trypsinised and counted by using a hemocytometer with 0.

2% trypan blue (Life Technologies). Around 5000 cells were seeded onto 96-well plates with DMEM medium supplemented with 10% FBS, 2mM L-glutamine, 100unitsml?1 streptomycin Carfilzomib (Life Technologies). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added at 24h time interval and signals were measured by a plate reader (BioRad Hercules, CA, USA). Cell invasion assay Cells were trypsinised and counted in 0.2% trypan blue (Life Technologies). Around 10000 cells were counted and resuspended in 100��l of serum-free DMEM medium.