Without removal of the transfection mixture, 500 ��l RPMI with 20% FBS was added. Cells were incubated for an additional 24 h. Transfected often cells were examined by cell proliferation assays and Western blotting. Immunohistochemistry. Immunohistochemistry for cyclin D1 and p21WAF1 was performed with a BenchMark XT automated stainer (Ventana Medical Systems, Tucson, AZ, USA). Tissue was incubated with anti-cyclin D1 antibody (SP4-R; Ventana) or anti-p21WAF1 antibody (sc-817; Santa Cruz Biotechnology) at 37��C for 28 min. Antigen was detected with an ultraView Universal diaminobenzidine (DAB) detection kit. Positive staining for cyclin D1 was defined as positive staining that exceeded 10% of the total cancer cell nuclei. Immunostaining for p21WAF1 is found in normal glandular epithelial cell nuclei of the gastric mucosa.

Loss of p21WAF1 was considered significant if staining for p21WAF1 was lost in more than 90% of cancer cell nuclei. Statistical analysis. The slopes of graphs and levels of apoptosis and proliferation were compared with linear mixed models incorporating R statistical software package nlme (http://cran.r-project.org/bin/windows/base/). The ��2 test, Pearson’s test, and Kendall’s Tau-b correlation analyses were conducted with IBM SPSS Statistics version 20.0 (IBM Corp., Somers, NY, USA) to evaluate correlations between EBV status and immunoexpression in gastric carcinoma tissues. A P value of less than 0.05 was considered statistically significant. RESULTS BARF1 protein is primarily secreted and weakly observed within SNU601 BARF1-transfected cells.

EBV-negative SNU601 gastric carcinoma cells were transfected with pCMV-Tag 2B/flag/BARF1 plasmid (SNU601 BARF1). BARF1-transfected cell clones were isolated by G418 selection. The recombinant plasmid pCMV-Tag 2B/flag/BARF1 was confirmed to be correct by restriction endonuclease digestion with BamHI/XhoI (Fig. 1A) and DNA sequencing (Fig. 2). Sequencing revealed SNU719 BARF1 to be virtually identical to the prototype B95-8 sequence, except for an A-to-G mutation at position 165710 (Fig. 2). Specific full-length transcripts of BARF1 were restricted to SNU719 cells (a naturally EBV-infected gastric carcinoma cell line) and SNU601 BARF1 cells, whereas BARF1 transcripts were not detected in SNU601 mock-transfected cells (here called SNU601 mock) (Fig. 1B). Fig 1 Generation of BARF1 stable transfectants in SNU601 cells. (A) The recombinant plasmid pCMV-Tag 2B/flag/BARF1 was digested with BamHI and XhoI. (B) BARF1 mRNA was detected in SNU719 (naturally EBV-infected gastric carcinoma cells) and SNU601 BARF1 (stably … Fig 2 Cloning of the EBV-carried BARF1 gene. Full-length BARF1 was amplified with primers F and R, which introduced BamHI and XhoI recognition sites, GSK-3 respectively.