5 months (range 5 to 53 months). Immunohistochemical analysis of tissue c-FLIP expression Sections (4 μm) were deparaffinized, rehydrated, immersed in 3% H2O2 for 10 min and microwaved at 750 W in citrate buffer (pH 6.0) for 15 BIBW2992 min. Tissue sections were then blocked for 20 min with normal rabbit serum and incubated overnight at 4°C with rabbit anti-human c-FLIP polyclonal antibodies, diluted 1:200(Abcan, UK). Incubation with PBS instead of the primary antibody served as a negative control. After washing

twice with PBS for 2 min each, immunostaining was performed using the standard S-P technique (Beijing Zhongshan Bio., China) and visualized with diaminobenzidine tetrahydrochloride solution. Staining was assessed blindly by one observer. A minimum of five click here randomly selected fields (200×) were examined, with a mean of 1500 cells counted throughout the whole ASP2215 cost section. The labeling index was defined as the percentage of neoplastic cells with clear cytoplasmic immunoreactivity of the total number of neoplastic cells counted. The threshold for c-FLIP positivity was 10%. The intensity of staining was scored as 0: achromatic, 1: light yellow, 2: yellow, 3: brown. Construction of RNAi vectors According the sequence of the c-FLIP mRNA, the siRNA oligonucleotides

were designed and synthesized to the targeted RNAi regions at 526~544, 1164~1182, 1305~1323 nt. Bgl II and Hind III sites were respectively generated at the 5′ and 3′ ends of the templates (as shown below). si-526: 5′-CCC GGAGCAGGGACAAGTTACA TTCAAGAGA TGTAACTTGTCCCTGCTCC TTTTTGGAAA-3′ (Forward); 5′-TTTCCAAAAA GGAGCAGGGACAAGTTACA TCTCTTGAA TGTAACTTGTCCCTGCTCC GGG-3′ (Back). si-1164: 5′-CCC GCGAGGGCTGTGCACAGTT TTCAAGAGA AACTGTGCACAGCCCTCGC TTTTTGGAAA-3′ (Forward); 5′-TTTCCAAAAA GCGAGGGCTGTGCACAGTT

TCTCTTGAA AACTGTGCACAGCCCTCGC GGG-3′ (Back). si-1305: 5′-CCC ACGCCCACTCCTGGATCTT TTCAAGAGA AAGATCCAGGAGTGGGCGT TTTTTGGAAA-3′ (Forward); 5′-TTTCCAAAAA ACGCCCACTCCTGGATCTT TCTCTTGAA AAGATCCAGGAGTGGGCGT GGG-3′ (Back). These above siRNA-encoding complementary single-stranded oligonucleotides were hybridized to give Bgl II- and Hind Lck III-compatible overhangs, and then ligated into pSuper (linked overnight at 16°C). After E. Coli, DH5α, transfected by the recombinant vectors, the positive clones were selected. With positive plasmids, the sequences were checked by sequencing a PCR-amplified region containing the oligonucleotides. The recombinant plasmids were named as pSuper-Si1, pSuper-Si2, pSuper-Si3 and pSuper-Neg(no target segment inserted), respectively. siRNA transfections HCC cell line, 7721, showed stronger staining intensity (results not shown), and was used as the target cell for the following study. 7721 cells were cultured in RPMI1640(Invitrogen, USA) containing 10% fetal bovine serum(FBS), and maintained in a humidified 37°C incubator with 5% CO2 by routine passage every 3 days or as needed.