Fig. 4

Sup-B15 cells are resistant to CpG 685 treatment and imatinib reverses the resistance A. The expression of phosphorylated P65, phosphorylated P38, C-MYC, phosphorylated p53 at ser46, and BAX have increased by 5 μg/mL CpG 685 treatment at the indicated time points in Sup-B15 cells, as determined by western blotting (left panel). However, the expression of JNK1 phosphorylation is decreased by CpG 685 treatment and ARF is almost undetectable. Densitometry of western blots was analyzed with ImageJ and is presented as a mean ± SD at each time point on the right panel. B Annexin-V/7AAD staining of apoptotic Sup-B15 cells with or without 0.1 mg/L imatinib pretreatment after culture in media with or without CpG 685 for 3 days. Combinational use of CpG 685 and imatinib to treat Sup-B15 cells reversed resistance to CpG 685 treatment alone. C The percentage of Annexin-V⁺ cells in Sup-B15 cells (fold) which were incubated alone or in the presence of CpG 685 with or without imatinib pretreatment for 3-day was analyzed by flow cytometry. Columns represent means of 3 independent experiments; bars represent SEM. *p < 0.05. D Western blot results showing that imatinib inhibits downstream molecules of BCR-ABL, such as phosphorylated STAT5, C-MYC, and BCL-XL. Active BAX was upregulated by imatinib and eventually induced apoptosis of Sup-B15 cells with CpG 685. Imatinib upregulates the phosphorylation of AKT at ser473. At the same time, CpG 685 dephosphorylates AKT at ser473 by upregulating PTEN, and ultimately reversed the drug resistance of imatinib, which caused by BCR-ABL1-independent AKT phosphorylation in Ph( +) B-ALL. (left panel) Results are presented as the mean ± SD on the right panel. *p < 0.05. E Immunofluorescence and laser scanning confocal microscopy showed that imatinib promotes the expression of BAX(6A7). BAX(6A7) staining represented the activated BAX. The combination therapy of CpG 685 and imatinib is a benefit for the BAX activation. F RT-PCR (down penal) and qRT-PCR (up penal) results of miR-21 and U6. MiR-21 was quantitated by normalizing ΔΔCt value over U6. CpG 685 downregulated miR-21 with or without imatinib in Sup-B15 cells. QRT-PCR results is presented as a mean ± SD at each time point on the right panel. Significant difference was accepted at *p < 0.05