Journal:

Article Title: Monocytes Treated with Human Immunodeficiency Virus Tat Kill Uninfected CD4 + Cells by a Tumor Necrosis Factor-Related Apoptosis-Induced Ligand-Mediated Mechanism

doi: 10.1128/JVI.77.12.6700-6708.2003

Figure Lengend Snippet: HIV Tat enters Jurkat cells but not U937 cells or monocytes. Jurkat cells, U937 cells, and monocytes were exposed to HIV Tat (100 ng/ml) for 0 min (lanes 1, 5, and 10), 30 min (lanes 2, 6, and 11), 1 h (lanes 3, 7, and 12), or 3 h (lanes 4, 8, and 13). After the indicated times, cells were collected, cell lysates were separated by SDS-10% PAGE, and cell proteins were transferred to nitrocellulose membranes and immunoblotted with the TR1 murine monoclonal antibody to Tat (57). Lanes 9 and 14 contain recombinant Tat protein.

Article Snippet: The human leukemia Jurkat T-cell line (clone E6.1; American Type Culture Collection) and promonocytic U937 cell line (no. CRL2367; American Type Culture Collection) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM l -glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml.

Techniques: Recombinant

Journal:

Article Title: Monocytes Treated with Human Immunodeficiency Virus Tat Kill Uninfected CD4 + Cells by a Tumor Necrosis Factor-Related Apoptosis-Induced Ligand-Mediated Mechanism

doi: 10.1128/JVI.77.12.6700-6708.2003

Figure Lengend Snippet: HIV Tat increases TRAIL expression in U937 cells and human monocytes, but Tat toxoid does not. (A) TRAIL expression increases with increasing doses of Tat protein. Monocytes (a) and CD4+ cells (b) were incubated with or without HIV Tat or Tat toxoid (10 to 1,000 ng/ml) at 37°C for 20 h, and 10 μl of cell lysate (6 × 104 cell equivalents) was separated by SDS-10% PAGE. Cell proteins were transferred to nitrocellulose membranes and immunoblotted with polyclonal anti-TRAIL (H-257; 1:1,000). Lanes (from left): 1, no HIV Tat protein; 2 to 5, cell extracts exposed to 10, 50, 100, and 1,000 ng of Tat/ml, respectively. Actin (antibody A-2668; Sigma) was an internal control. Similar results were obtained with fresh human monocytes and with the U937 cell line. TRAIL production in fresh human T cells or in Jurkat cells was not stimulated by exposure to Tat. (B) HIV Tat induces the transcription of TRAIL mRNA in U937 cells and monocytes. Total RNA from U937 cells, monocytes, or CD4+ cells was isolated and subjected to reverse transcription-PCR. β-Actin mRNA was amplified over 25 cycles and TRAIL mRNA was amplified over 35 cycles of PCR. Transcription of control β-actin or TRAIL/APO2L mRNA in U937 cells is shown after 0, 2, 6, 12, or 24 h of incubation in the presence of HIV Tat (100 ng/ml).

Article Snippet: The human leukemia Jurkat T-cell line (clone E6.1; American Type Culture Collection) and promonocytic U937 cell line (no. CRL2367; American Type Culture Collection) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM l -glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml.

Techniques: Expressing, Incubation, Isolation, Amplification

Journal:

Article Title: Monocytes Treated with Human Immunodeficiency Virus Tat Kill Uninfected CD4 + Cells by a Tumor Necrosis Factor-Related Apoptosis-Induced Ligand-Mediated Mechanism

doi: 10.1128/JVI.77.12.6700-6708.2003

Figure Lengend Snippet: HIV Tat only slightly increases membrane-associated TRAIL expression in U937 cells and monocytes. U937 cells (A), human monocytes (B), Jurkat cells (C), and primary CD4+ cells (D) were stimulated with HIV Tat (100 ng/ml) for 20 h, stained with PE-conjugated mouse anti-human TRAIL or mouse IgG1 control monoclonal antibody as described in Materials and Methods, and then subjected to flow cytometry. The histograms shown are representative of three independent experiments.

Article Snippet: The human leukemia Jurkat T-cell line (clone E6.1; American Type Culture Collection) and promonocytic U937 cell line (no. CRL2367; American Type Culture Collection) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM l -glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml.

Techniques: Expressing, Staining, Flow Cytometry

Journal:

Article Title: Monocytes Treated with Human Immunodeficiency Virus Tat Kill Uninfected CD4 + Cells by a Tumor Necrosis Factor-Related Apoptosis-Induced Ligand-Mediated Mechanism

doi: 10.1128/JVI.77.12.6700-6708.2003

Figure Lengend Snippet: HIV Tat increases secretion of soluble TRAIL in media of U937 cells and monocytes. For this experiment, 3 × 106 U937 cells (A), human monocytes (B), and CD4+ cells (C) in 100 μl of medium were incubated with or without HIV Tat (10 to 1,000 ng/ml) at 37°C for 20 h, and then 20 μl of cell medium was separated by SDS-10% PAGE. Proteins were transferred to nitrocellulose membrane and incubated with polyclonal anti-TRAIL (H-257; 1:1,000). Lanes (from left), 1, no HIV Tat protein; 2 to 5, extracellular media exposed to 10 to 1,000 ng of Tat/ml. TRAIL secretion could not be detected from T-lymphocyte cultures after 20 h.

Article Snippet: The human leukemia Jurkat T-cell line (clone E6.1; American Type Culture Collection) and promonocytic U937 cell line (no. CRL2367; American Type Culture Collection) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM l -glutamine, 100 U of penicillin/ml, and 100 μg of streptomycin/ml.

Techniques: Incubation