Fig. 3

C-MYC-mediated BAX activation leads to CpG 685-induced caspase-independent apoptosis. A BLIN-1 cells with or without 25 μM 10058-F4 pretreatment were cultured in media with or without CpG 685 for 24 h. The expression and localization of Mito Tracker (red) and BAX(6A7) (green) were detected and evaluated by immunofluorescence and laser scanning confocal microscopy. CpG 685 can promote the expression of BAX(6A7) when C-MYC is activated and participate in the enrichment of BAX(6A7) to mitochondria. That is, 10058-F4 can block the activation of BAX by CpG 685. All images are analyzed by ImageJ software. B BAI1 is an inhibitor of BAX conformational activation. QVD represents Quinoline-Val-Asp-Difluorophenoxymethylketone (Q-VD-OPh) is a kind of effective pan-caspase inhibitor, which can prevent the cleavage of PARP mediated by caspase cascade. Apoptotic and viable BLIN-1 cells were determined by Annexin V/7AAD staining. Kinetic analysis showed that 0.5 μM BAI1 pretreatment inhibited CpG 685 induced Annexin V-positive apoptotic BLIN-1 cell number, while 80 μM QVD pretreatment did not block CpG 685 induced BLIN-1 apoptosis (left panel). The percentage of Annexin-V⁺ cells (fold) was analyzed by flow cytometry (right panel). Columns represent means of 3 independent experiments; bars represent SEM. *p < 0.05, **p < 0.01. The red line represents onefold. C Viable B-ALL cells cultured in media with or without 5 μg/mL CpG 685 after 3 day. Cells were examined by annexin-V/7AAD staining. The percentage of annexin V-positive B-ALL cells represent the apoptotic cells. CpG 685 induces apoptosis in RS4;11 cells, while NALM-6 and Sup-B15 cells are resistant to CpG 685 treatment (left panel). The percentage of Annexin-V⁺ cells (fold) was analyzed by flow cytometry (right panel). Columns represent means of 3 independent experiments; bars represent SEM. *p < 0.05. The red line represents onefold. D By comparing the protein expression levels of B-ALL cells with or without CpG 685, western blot results showed that three Ph( − ) B-ALL cells (BLIN-1, RS4;11, and NALM-6) overexpressed C-MYC compared to levels in Sup-B15 cells. Although CpG 685 can activate P38/P53 signaling pathway in BLIN-1, RS4;11, and NALM-6 cells, it cannot induce apoptosis in NALM-6 due to the loss of BAX expression. Besides, CpG 685 also cannot promote PARP cleavage in Ph( +) B-ALL cell (Sup-B15). Columns in the right panel represent means of at least 3 independent experiments; bars represent SD. Significant differences were accepted at *p < 0.05 against the group with no CpG 685 on each cell line. **p < 0.01. E With IgG as the control and input as the benchmark, ChIP-qPCR was used to determine the binding of C-MYC to the E-box region of the bax promoter. BLIN-1 and RS4;11 cells stimulated by CpG 685 for 12 h can promote the combination of C-MYC and E-box 1, 2, 3 and 4 C-MYC could not directly bind to the BAX promoter on Sup-B15 cells. All data are shown with mean ± SEM. Significant differences were accepted at *p < 0.05. **p < 0.01