About 4 × 106 TAT-expressing QGY-7703 cells or

control Vec-7703 cells were injected subcutaneously into the right and left hind legs of 4-week-old nude mice (10 mice for TAT-c2 and 10 mice for TAT-c3 cells), respectively. Tumor formation in nude mice was monitored over a 4-week period. Details are described in the Supporting Materials and Methods. TAT-transfected and vector-transfected QGY-7703 cells were treated with straurosporine (STS; 1 μM) for 4 hours. Morphological changes in the nuclear chromatin undergoing apoptosis were detected by terminal deoxynucleotidyl TUNEL assay according to the manufacturer’s protocol (Roche, Mannheim, Germany). Triplicate independent experiments were performed. Loss of mitochondrial membrane potential (ΔΨm), indicative of apoptosis, was detected using the MitoPT JC-1 detection kit (Immunochemistry Technologies, Bloomington, MN) according to the manufacturer’s protocol. Briefly, cells were check details cultured click here on the coverslips to 80% confluence in a 6-well plate. After STS treatment, cells were washed twice with phosphate-buffered saline (PBS) and then incubated with the ΔΨm-sensitive dyes JC-1 at 37°C for 15 minutes. Images

were captured using a Leica DMRA fluorescence microscope (Rueil-Malmaison, France). Details are described in the Supporting Materials and Methods. Western blot analyses were performed with the standard method with antibodies to TAT (Uscnlife Science & Technology, Wuhan, China), PAPR (Boster FAD Biotechnology, Wuhan, China), cytochrome-c (Cyt-c), caspase-9, caspase-8, and β-actin (Cell Signaling Technology, Danvers, MA). Statistical analysis was performed with the SPSS standard version 13.0 (Chicago, IL). The statistical significance of the correlations between TAT expression and loss of TAT allele, promoter methylation, as well as consistency between CGH and qPCR were assessed by a chi-square test. Results expressed as mean ± SD were analyzed using Student’s t test. Differences were considered significant when P was less than 0.05. In our previous CGH study, deletion of 16q was detected in 35/50 (70%) of primary

HCCs.3 To accurately estimate the loss of TAT allele in HCC, qPCR was used to compare DNA copy-number ratio between tumor and paired nontumor tissues in 50 HCC cases tested by CGH. Using a cutoff value of ≤0.5 to define DNA copy-loss, loss of TAT allele was detected in 27/50 (54%) of HCCs (Table 1). Compared with CGH results, 26/27 cases with TAT allele loss detected by qPCR was also detected by CGH (Fig. 1A), suggesting that the qPCR result was reliable. Loss of TAT allele was not detected in 9/35 cases with 16q loss detected by CGH, implying that the frequency of the loss of the TAT allele was lower compared with the CGH result. Statistical analysis confirmed the consistency between the CGH and qPCR results (Fig. 1B, R = 0.528; P < 0.0001).