.. BARF1 protein selleck chemical expressed in SNU601 BARF1 cells was detected as a 23- to 26-kDa band on Western blots with an anti-Flag antibody and the 4A6 anti-BARF1 antibody (Fig. 1C). BARF1 protein was secreted and was observed mainly in culture supernatants and only marginally detectable in cell lysates of SNU601 BARF1. To confirm that BARF1 is secreted via the classical pathway, we first examined BARF1 protein expression in cell culture supernatants treated with brefeldin A, which hinders export from distal Golgi compartments to the cell surface (Fig. 1D and andE).E). BARF1 was detected in cell lysates upon treatment with brefeldin A, and BARF1 secretion was almost completely inhibited (Fig. 1D). These results indicate that BARF1 is a classically secreted protein.

Immunofluorescence analysis showed that brefeldin A blocked secretion and caused retention of the BARF1 protein in the cytoplasm and nucleus (Fig. 1E). BARF1-expressing SNU601 cells show increased proliferation. SNU601 BARF1 cells showed higher proliferation than did SNU601 mock cells (P < 0.05) (Fig. 3B). There were no statistical differences in apoptosis, invasion, or migration between SNU601 BARF1 and SNU601 mock cells (Fig. 3A, ,C,C, and andD).D). The effect of BARF1 knockdown on cell proliferation was investigated in SNU719 cells, which express high endogenous levels of BARF1. SNU719 cells, which are naturally infected with EBV, were transfected with different siRNA oligonucleotides against BARF1 type 1, 2, or 3. Knockdown was verified by RT-PCR. BARF1 mRNA expression was markedly inhibited by the siRNA against BARF1 type 3 (Fig.

4A). Interestingly, we observed a significant reduction in the growth rate of SNU719 cells transfected with siRNA against BARF1 type 3 compared to cells transfected with scrambled siRNA (P < 0.05) (Fig. 4B). Fig 3 Biological properties of BARF1-expressing gastric carcinoma cells. (A) Apoptosis assay. Plots of flow cytometric data indicated no difference in apoptosis between SNU601 BARF1 and SNU601 mock. The percentage of apoptotic cells was determined by calculating ... Fig 4 Analysis of SNU719 (naturally EBV-infected gastric carcinoma cells) transfected with siRNA against BARF1. (A) BARF1 expression was almost completely inhibited upon transfection of 20 ��M siRNA against BARF1 type 3. (B) Cell proliferation was lower ...

BARF1-induced NF-��B RelA increment is associated with cyclin D1 and p21WAF1 expression. In this study, we focused on the IRAK1/I��B��/NF-��B/cyclin D1 Brefeldin_A pathway (31, 33). We observed increased expression of the NF-��B RelA protein in nuclear extracts of SNU601 BARF1 cells compared with SNU601 mock cells (Fig. 5). Increased nuclear expression of NF-��B RelA correlated with increased expression of cyclin D1 and reduced expression of p21WAF1. Interestingly, expression of NF-��B RelA did not correlate with levels of the NF-��B-modulator IRAK1.