Ab8805

Paraffin-embedded HCC tissue sections (4-μm) on poly-1-lysine-coated slides were deparaffinized and rinsed with 10 mM Tris-HCl (pH 7.4) and 150 mM sodium chloride. Peroxidase was quenched with methanol and 3% hydrogen peroxide. Slides were then placed in 10 mM citrate buffer (pH 6.0) at 100 °C for 20 min in a pressurized heating chamber. After incubation with 1 : 200 dilution of p-Akt1/2/3 (Thr 308)-R antibody (ab8805, Abcam, Cambridge, UK) and with 1 : 100 dilution of CIP2A antibody (ab84547, Abcam) for 1 h at room temperature, slides were thoroughly washed three times with PBS. Bound antibodies were detected using the EnVision Detection Systems Peroxidase/DAB, Rabbit/Mouse kit (Dako, Glostrup, Denmark). The slides were then counterstained with hematoxylin. Paraffin-embedded sections of mouse kidney tissue and human colon carcinoma were used as positive controls for p-Akt1/2/3 and CIP-2A, respectively, as described in the datasheet from the manufacturer. Negative controls had the primary antibody replaced by PBS. The expression of p-Akt1/2/3 and CIP-2A was assessed semiquantitatively based on the intensity of staining by a board certified pathologist. The intensity of staining was scored as negative, weak, moderate and strong.
This study was approved by the ethics committee of the Institutional Review Board of Changhua Christian Hospital. All informed consents from sample donors were in accordance with the Declaration of Helsinki and were obtained at the time of their donation.

Cell lines and culture. Bel7402, Bel7404, and SMMC7721 cells (human hepatocellular carcinoma cell lines) were cultured in RPMI-1640 (Thermo Fisher Scientific, Shanghai, China) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% fetal bovine serum. The cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cultures were grown at 37°C in a humidified atmosphere containing 5% carbon dioxide13 (link). The OXA (oxaliplatin)-resistant cells, termed Bel7404-OR, were established by incubating Bel7404 cells with OXA13 (link).
Antibodies and reagents. The antibodies used in this study included an antibody against clusterin (#42143), which was obtained from Cell Signaling Technology (Danvers, MA, USA), and antibodies against GADD45a (ab180768), AKT1 (ab18206), AKT (ab8805), Bcl-2 (ab32124) and Bax (ab32503), which were obtained from Abcam (Cambridge, UK). The PI3-kinase inhibitor (LY294002) (ab120243) was purchased from Abcam (Cambridge, UK).
IHC and histopathological analyses. Human hepatocellular carcinoma tissue arrays were obtained from Shanghai Biochip Company Ltd. (Shanghai, China). HCC tissues were obtained from 105 HCC patients undergoing complete surgical resection between January 2007 and November 2009. All patients were followed up until September 2013. None of the patients received radiotherapy or chemotherapy before surgery, and none of them had multiple cancers in other organs.
The tissue microarray was deparaffinized, rehydrated through graded alcohol series, washed with Tris-buffered saline, and processed using a streptavidin-biotin-peroxidase complex method. The tissue microarray was incubated with mouse anti-Clusterin (1:200, Cell Signaling Technology Danvers, MA, USA) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody. Next, the tissue microarray was assessed by two pathologists who were blinded to the clinical and pathologic information. The staining intensity of the tumor tissues was scored as 0(negative), 1+ (weak), 2+ (moderate) or 3+ (strong), while the staining extent was scored as 0 (<10%), 1+ (10%-25%), 2+ (26%-50%) or 3+ (>50%)15 (link), 16 (link). The product of staining intensity by the staining extent was defined as the final staining score which was classified as negativity (score 0-3) and positivity (score 4-9)17 (link).
Plasmid construction and transfection, cell viability and apoptosis assays. Bel7404 cells stably transfected with the pIRES2-EGFP/sCLU and pIRES2-EGFP vectors were termed Bel7404-sCLU^high and Bel7404-vec, respectively. Bel7402 or SMMC7721 cells stably transfected with sCLU shRNA (CLU-4) or Sc vectors were termed Bel7402-sCLU^low, Bel7402-vec, SMMC7721-sCLU^low or SMMC7721-vec. The Gadd45a expression vector was constructed and termed pcDNA3.1-Gadd45a-His. The plasmids were transfected into Bel7402 and SMMC7721 cells and were termed as Bel7402-Gadd45a and SMMC7721-Gadd45a, respectively. All these methods were described previously13 (link), 14 (link).
Western blot analysis for protein expression. Whole proteins from cultured HCC cells were extracted using a protein extraction kit (Beyotime, Shanghai, China). In total, 50 µg of proteins was resolved on an 8% SDS-PAGE gel and was transferred to a PVDF membrane (Millipore). Non-specific binding sites in the membranes were blocked with 1% bovine serum albumin in TBST buffer for 2 h at room temperature before the addition of primary antibodies. Appropriate anti-rabbit, anti-mouse and anti-goat antibodies were used as secondary antibodies for 2 h at 37°C. Protein bands were detected by using an ECL detection kit (Millipore, MA, USA) and protein expression was determined using Quantity One software and was normalized to GAPDH.
Statistical analysis. All data were expressed as the mean ± standard deviation (SD) and were analyzed by SPSS 17.0 software. Quantitative data were analyzed using one-way ANOVA. Survival curves were evaluated using the Kaplan-Meier method and were compared using the log-rank test. A value of p <0.05 was considered significant.