Evidentially, treatment with 1 μM CpG-ODN for 8 h reduced the frequency of FasL-expressing HepG2 cells to 28% and treatment for 24 h decreased the frequency of FasL-expressing HepG2 cells to near 10%. Apparently, treatment with CpG-ODN inhibited the expression of FasL in HepG2 cells in a dose- and time-dependent manner. Figure 1 Treatment with CpG-ODN inhibited the expression of FasL in HepG2 cells in a dose- and time-dependent manner. (A) Dose effect. HepG2 cells were treated with different concentrations of CpG-ODN for 48 h. (B) Time
effect. HepG2 cells were treated with 1 μM CpG-ODN for the indicated time periods. The cells were harvested, and the frequency of FasL-positive cells was determined by FACS analysis. INCB28060 in vitro Data are expressed as mean% ± SEM of each group of the cells from four independent GSK2245840 experiments. *p < 0.05 vs. controls. Effect of CpG-ODN on the Fas expression in Jurkat cells Next,
we tested whether treatment with CpG-ODN could modulate the expression of Fas in Jurkat cells. Jurkat cells were treated with 1 μM CpG-ODN for 24 h. The cells were harvested and the relative levels of Fas mRNA transcripts to control GAPDH were determined by quantitative RT-PCR (Figure 2A). Clearly, the relative levels of Fas mRNA transcripts in the CpG-ODN-treated Jurkat cells were reduced to 65%, as compared with that of unmanipulated controls. Furthermore, CHIR98014 the expression of Fas in Jurkat cells was also examined by flow cytometry analysis. The frequency of Fas-expressing Jurkat cells was significantly reduced from 54% ± 2% to 35% ± 1% (Figure 2B). Therefore, CpG-ODN treatment down-regulated the Fas mRNA transcription and protein expression in Jurkat cells in vitro. Figure 2 Treatment with CpG-ODN inhibited the expression of Fas in Jurkat cells. Jurkat cells
were treated with 1 μM CpG-ODN for 24 h, and the cells were collected. PI-1840 The intracellular expression of Fas was examined by qRT-PCR (A) and FCM (B). Data are expressed as mean% ± SEM of each group of the cells from four separate experiments. *p < 0.05 vs. the controls. Effect of CpG-ODN on the HepG2-mediated Jurkat cell apoptosis Engagement of Fas on the cell membrane by FasL can trigger cell apoptosis. Given that CpG-ODN treatment down-regulated the expression of FasL in HepG2 cells and Fas in Jurkat cells, it is possible that CpG-ODN may modulate the HepG2 cell-mediated Jurkat cell apoptosis. Accordingly, we first treated HepG2 and Jurkat cells with 1 μM CpG-PDN or anti-FasL NOK-2 antibody for 24 h for the preparation of effector and target cells, respectively. Next, we co-cultured the unmanipulated HepG2 and Jurkat cells (positive controls), the NOK-2-treated HepG2 and untreated Jurkat cells, the untreated HepG2 and the NOK-2-treated Jurkat cells, the CpG-ODN-treated HepG2 and untreated Jurkat cells, and the untreated HepG2 and the CpG-ODN-treated Jurkat cells for 24, respectively.