Table 1.
Sequences of quantitative real-time polymerase chain reaction (RT-qPCR) primers.
Fig 1.
Differential expression of apoptosis regulating factors in latently HIV-1-infected cells.
(A) The protein expression levels of pro-apoptotic and anti-apoptotic factors, caspase-3, -7, and -9; and PARP were compared between latently HIV-1-infected (ACH2, J1.1, and U1) cells and the parent cells (A3.01, Jurkat, and U937) by Western blotting analysis using the indicated antibodies. (B) Comparison of the protein expression levels of FAS, FASL, MLKL, RIP, and caspase-9 associated with the extrinsic apoptosis pathway in latently HIV-1-infected and non-infected cells. (C) The protein expression level of LC3B is related to the autophagy pathway in these cells. Housekeeping β-actin was used as a loading control.
Fig 2.
ABT-263 induced apoptosis of cells in latently HIV-1-infected cells.
(A) The cells were treated with 1 μM BAX agonists (BTSA1 and SMBA1), BCL-2 antagonists (ABT-263, AT-101, and GX15-070), and IAP antagonists (AT-406, Birinapant [BP], and LCL-161) for 24 h. The sub-G1 population (dead cells) was determined by flow cytometry after PI staining. (B) Results of three independent sub-G1experients are represented as graphical data expressed as means ± SD (n = 3). *p < 0.05, compared with A3.01 cells treated with the same agent. (C) The cells were treated with indicated agents as described in (A). Early and late phases of apoptotic cells were analyzed by flow cytometry using annexin V-FITC/PI staining.
Fig 3.
ABT-263 induced apoptosis executor proteins in latently HIV-1-infected cells.
(A) A3.01 and ACH2 cells were treated with indicated apoptosis inducing agents at different concentrations (0.1, 0.5, 1, and 5 μM) for 24 h, and cell viabilities of these cells were determined using the MTT assay. Data are represented as the relative mean value ± SD (n = 3) compared with DMSO control. *p < 0.05, **p < 0.01, compared with A3.01 cells treated with the same conditions. (B) A3.01 and latently infected ACH2 cells treated with ABT-263 (0, 1, and 5 μΜ) for 24 h. At 24 h after treatment, levels of cleavage of PARP, caspase-3, and -9 were analyzed by Western blotting using the indicated antibodies. β-actin was used as a loading control.
Fig 4.
Synergistic effect of the combination of ABT-263 on the selective death of latently HIV-1-infected cells.
(A) The cells were treated with ABT-263 (1 μM) or its combination with BTSA1 (1 μM), GX15-070 (1 μM), and BP (1 μM), respectively. At 24 h after treatment, the sub-G1 population of cells was determined by flow cytometry after PI staining. (B) The cells were treated with ABT-263 (1 μM) or its combinations (1 μM) for 24 h. Subsequently, early and late phase of apoptotic cells were analyzed by flow cytometry using annexin V-FITC/PI staining. (C) A3.01 and ACH2 cells were treated with ABT-263 (1 μM) and its combination with GX-15-070 (1 μM). At 24 h after treatment, expression of proteins was analyzed by Western blotting using the indicated antibodies and β-actin as a loading control.
Fig 5.
Change of mitochondrial membrane potential and cytochrome C release.
(A) The cells were treated with ABT-263 (1 μM) or its combination with BTSA1 (1 μM), GX15-070 (1 μM), and BP (1 μM), respectively. At 24 h after treatment, the mitochondrial membrane potential of these cells was determined by flow cytometry after JC-1 staining. Red and green cells indicate high (PE) and low (FITC) mitochondrial membrane potential, respectively. (B) The cells were treated with ABT-263 and its combinations, and the cells containing low mitochondrial membrane potential (FITC) after staining with JC-1 were visualized using fluorescence microscopy. (C) The cells were treated with ABT-263 and its combinations for 12 h, and the mitochondria and cytosol were fractionated from those cells. The levels of cytochrome C were analyzed by Western blotting. COX IV and β-actin were used as loading controls. Cyto; Cytoplasm, Mito; Mitochondria.
Fig 6.
Apoptosis effects of apoptosis inducing agents on acutely HIV-1-infected cells.
(A) A3.01 cells were infected with HIV-1NL4-3 at an MOI of 0.1. At 24 h after infection, the expression levels of proteins were measured by Western blotting using the indicated antibodies. (B) Cells infected acutely with HIV-1NL4-3 were treated with BTSA1 (1 μM), ABT-263 (1 μM), GX15-070 (1 μM), and BP (1 μM), respectively. At 24 h after treatment, the sub-G1 population of cells was determined by flow cytometry after PI staining.
Fig 7.
Selective killing effects of ABT-263 and its combination in a primary HIV-1 infection model.
(A) Schematic process for establishment of primary HIV-1 infection model. The naïve CD4+ T cells sorted magnetically from human PBMCs were activated and expaned with treatment of anti-CD3/CD28 antibodies and cytokines (IL-4, IL-12, TGF-B1, and IL-2) for 7 days, that led to major differentiation of human memory and effector CD4+ T cells. After the differentiation, the cell mixtures were infected with HIV-1NL4-3 at an MOI of 1 for 7 days; the cells were then treated with apoptosis-inducing agents for 1 day. To determine the latently infected cells, the cells were reactivated with anti-CD3/CD28 antibodies and measured by flow cytometry. (B) The isolated naïve CD4+ T cells from human PBMCs (upper panel, CD45RO-RA+, 0 day) and differentiated CD4+ T cells (lower panel, CD45RO+RA-, 7 days) were analyzed by flow cytometry. (C) The differentiated CD4+ T cell mixture was infected with HIV-1NL4-3 for 7 days; the cells were then treated with ABT-263 (0.05 μM), BTSA1 (0.5 μM), GX15-070 (0.5 μM), and combinations of ABT-263 for 24 h and were subsequently reactivated with anti-CD3/CD28 antibodies. The populations of HIV-1 p24+ cells and early and late phases of apoptotic cells were analyzed by flow cytometry using HIV-1 p24 and annexin V-FITC/PI staining.