Figure 5. Inhibition of autophagy enhances anticancer activity of IFN1@. (A and B) K562 cells were treated with IFN1@ (1000 U/ml) for 48 h. Apoptosis was analyzed by measuring annexin V-positive cells by flow cytometry (A). In parallel, CASP3 activation was assayed (B). n = 3, *p < 0.05 vs. control shRNA group. (C) Indicated HL-60 and Jurkat cells were treated with IFN1@ (1000 U/ml) for 48 h. Apoptosis was analyzed by measuring annexin V-positive cells by flow cytometry. n = 3, *p < 0.05 vs. control shRNA group. (D) CD34⁺ and CD34^- cells isolated from bone marrow mononuclear cells of CML patients by CD34 MicroBead Kit (Miltenyi Biotec) or imatinib-resistant K562 cells were transfected with BECN1 shRNA or control shRNA for 48 h, and then treated with IFN1@ (1000 U/ml) for 48 h. Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, and apoptosis was analyzed by measuring annexin V-positive cells by flow cytometry. In parallel, mRNA levels of BECN1 were analyzed by real-time PCR. n = 3, *p < 0.05 vs. control shRNA group. (E) Western blot analysis of LC3 processing by autophagy in the presence or absence of chloroquine (200 μM) after IFN1@ (1000 U/ml) treatment for 48 h in K562 cells. (F and G) K562 cells were treated with IFN1@ (1000 U/ml) for 48 h in the presence or absence of chloroquine (50–200 μM). Apoptosis was analyzed by measuring annexin V-positive cells by flow cytometry (F). In parallel, CASP3 activation was assayed (G). n = 3, *p < 0.05 vs. the group without chloroquine treatment.