1 (-) vector (Invitrogen) PF-02341066 clinical trial to generate pcDNA3.1 (-)-PDCD4 construct. The recombinant was identified by double digestion with restriction enzymes and DNA sequencing was performed by Shanghai Sangon Biological Engineering Technology and Service Co. Ltd (Sangon). Cell transfection In order to study the effects of PDCD4,

we transfected the pcDNA3.1 (-)-PDCD4 plasmid into MHCC-97H cells which was shown to express lowest level of PDCD4. Cells were grown to 70% confluence in 6-well plates, pcDNA3.1 (-)-PDCD4 plasmid (2 μg per well) was transfected by LipofectAMINE 2000 (Invitrogen) according to the manufacturer’s instructions. The empty vector pcDNA3.1 (-) was also transfected as a control. The parental cells without transfection were considered to be another control group. Stable clones were generated by selection in complete culture medium containing 400 μg/mL G418 48 h later. Western blot analysis was taken to identify the effectiveness of the PDCD4 transfection[20].

Western blot analysis Western blot analyses were performed to detect the expression of PDCD4, to identify the effectiveness of the PDCD4 gene transfection and to analyze the expression of MTA1 after PDCD4 transfection. HCC cells BAY 73-4506 grown to 70–90% confluence were washed with ice-cold PBS for two times and then collected by scraping. The cell pellets were homogenized in extraction buffer (50 mM Tris-HCl, 0.1% SDS, 150 mM NaCl, 100 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1% Nonidet P-40, and 0.5% sodium orthovanadate), then incubated at 4°C for 30 min and centrifuged 20 min at 12,000 g/min.

The total proteins (50 μg per lane) were resolved in 10% SDS-polycrylamide gels, and then transferred onto nitrocellulose membrane (0.45 μm, Millipore, Bedford, MA, USA) in 25 mM Tris-base, 190 mM glycine, and 20% methanol using a semi-dry blotter. Following blocking with 10% nonfat milk and 0.1% Tween20 in TBS for 2 h, the membranes were incubated with anti-PDCD4(Santa Cruz Biotechnology, Santa Cruz, California, FAD USA, 1:200 for gene expression and 1:2000 for identification of transfection), anti-MTA1(Santa Cruz Biotechnology, Santa Cruz, California, USA, 1:500) or anti-β-actin (Jingmei Biotech, Shenzhen, China, 1:2000), respectively, at 4°C overnight. After binding of horseradish peroxidase (HRP)-coupled goat anti-mouse or goat anti-rabbit IgG (Jingmei Biotech, Shenzhen, China,1:5000) at room temperature for 2 h, antigens were visualized by enhanced chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, California, USA,) Bands corresponding to different proteins were scanned and the respective areas and IOD were determined using Image-Pro Plus 6.0. The relative densities were calculated by normalizing the IOD of each blot with that of β-actin [21].