Fig. 2

CpG 685-induced apoptosis of BLIN-1 cells is P38/P53/BAX and JNK/C-MYC signaling pathway dependent. A CpG 685 activates downstream molecules of the TLR9 signaling pathway in BLIN-1 cells. Phosphorylated P65, phosphorylated P38, phosphorylated JNK, C-MYC, ARF, P53, phosphorylated p53 at ser46, BAX, BAK, and PARP cleavage in BLIN-1 cells cultured with or without 5 μg/mL CpG 685 were examined by western blotting at the indicated time points (left panel). All data shown are representative of 3 independent experiments (right panel), mean ± SD. B BLIN-1 cells with or without SB203580 pretreatment were cultured in media with or without CpG 685 for 24 h. Phosphorylated p53 at ser46, BAX expression, and PARP cleavage were reversed by SB203580 pretreatment of BLIN-1 cells under CpG 685 treatment (left panel). All data shown are representative of 3 independent experiments (right panel), mean ± SD. *p < 0.05. C BLIN-1 cells with or without SP600125 pretreatment were cultured in media with or without CpG 685 for 24 h. The activations of C-MYC and PARP cleavage of BLIN-1 cells under CpG 685 treatment can be reversed by SP600125 pretreatment (left panel). All data shown are representative of 3 independent experiments (right panel), mean ± SD. *p < 0.05. D JNK inhibition by SP600125 enabled BLIN-1 to overcome CpG 685 induced apoptosis. Cells were incubated alone or in the presence of CpG 685 with or without 40 μM SP600125 pretreatment for 72 h. Cells were then harvested and labeled with antibodies against Annexin-V and 7AAD to define the apoptotic cells. E C-MYC inhibition by 10058-F4 enabled BLIN-1 to overcome CpG 685 induced apoptosis. Cells were incubated alone or in the presence of CpG 685 with or without 25 μM 10058-F4 pretreatment for 72 h. Cells were then harvested and labeled with antibodies against Annexin-V and 7AAD to define the apoptotic cells. F BLIN-1 cells with different doses of 10058-F4 pretreatment were cultured in media with or without CpG 685 for 24 h. ARF expression, P53 expression, activation of phosphorylated p53 at ser46, BAX expression, and PARP cleavage were reversed by 25 μM 10058-F4 pretreatment before CpG 685 stimulation (left panel). Densitometry of western blots was analyzed with ImageJ and is presented as a mean ± SD at each dose point on the right panel. Columns represent means of at least 3 independent experiments. Significant differences were accepted at *p < 0.05 against the group with no CpG 685 at each dose of inhibitor pretreatment and calculated according to the two-tailed Student’s t-test