Journal: AIDS Research and Human Retroviruses

Article Title: Short Communication: The Broad-Spectrum Histone Deacetylase Inhibitors Vorinostat and Panobinostat Activate Latent HIV in CD4 + T Cells In Part Through Phosphorylation of the T-Loop of the CDK9 Subunit of P-TEFb

doi: 10.1089/aid.2015.0347

Figure Lengend Snippet: Vorinostat (SAHA) and panobinostat induce CDK9 Thr-186 T-loop phosphorylation in resting CD4+ T cells. Resting CD4+ T cells were isolated by negative selection from peripheral blood of healthy donors by the Rosettesep CD4+ cells isolation kit (STEMCELL Technologies, Inc.). Flow cytometry analysis confirmed that the CD4+ cell populations were routinely >98% (data not shown). Cells were treated with various concentrations of vorinostat, panobinostat, tacedinaline, or romidepsin for 24 h and lysates were prepared in EBCD buffer [50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.5% Nonidet P-40, 5 mM dithiothreitol, 4 mM MgCl2 buffer containing protease inhibitor cocktail] and analyzed for the expression of indicated proteins in immunoblots. Experiments were repeated at least three times and representative figures are shown. CDK9, cyclin-dependent kinase 9.

Article Snippet: Resting CD4 + T cells were isolated by negative selection from peripheral blood of healthy donors by the Rosettesep CD4 + cells isolation kit (STEMCELL Technologies, Inc.).

Techniques: Phospho-proteomics, Isolation, Selection, Flow Cytometry, Protease Inhibitor, Expressing, Western Blot

Journal: AIDS Research and Human Retroviruses

Article Title: Short Communication: The Broad-Spectrum Histone Deacetylase Inhibitors Vorinostat and Panobinostat Activate Latent HIV in CD4 + T Cells In Part Through Phosphorylation of the T-Loop of the CDK9 Subunit of P-TEFb

doi: 10.1089/aid.2015.0347

Figure Lengend Snippet: Time course of CDK9 T-loop phosphorylation in HDACi-treated CD4+ T cells. CD4+ T cells prepared as described in Figure 1 were treated with either vorinostat or panobinostat for the indicated time periods. Cell lysates were prepared at the indicated times as described in Figure 1 and lysates were examined in immunoblots. HDACi, histone deacetylase inhibitor.

Article Snippet: Resting CD4 + T cells were isolated by negative selection from peripheral blood of healthy donors by the Rosettesep CD4 + cells isolation kit (STEMCELL Technologies, Inc.).

Techniques: Phospho-proteomics, Western Blot, Histone Deacetylase Assay

Journal: AIDS Research and Human Retroviruses

Article Title: Short Communication: The Broad-Spectrum Histone Deacetylase Inhibitors Vorinostat and Panobinostat Activate Latent HIV in CD4 + T Cells In Part Through Phosphorylation of the T-Loop of the CDK9 Subunit of P-TEFb

doi: 10.1089/aid.2015.0347

Figure Lengend Snippet: Vorinostat and panobinostat activate HIV-1 in CCL19-mediated HIV-1 latency model in resting CD4+ T cells. (A) Purified resting CD4+ T cells were cultured for 3 days in the presence of CCL19 (10 nM; PeproTech, NJ) or were left inactivated. As a positive control, CD4+ cells were treated with PMA+ionomycin for 16 h. Cell lysates were prepared and examined in immunoblots. (B) Resting CD4+ T cells were cultured for 3 days in the presence of CCL19 and then infected for 5 days with the HIV-1 NL4.3-Luc reporter virus, which contains a deletion in the nef gene (Δnef) and replaced with luciferase gene (HIV-1 NL4.3 Δnef luc). Cells were treated with vorinostat, panobinostat, or tacedinaline for 24 h at the indicated concentrations. Cell lysates were prepared and examined in immunoblots (B), and luciferase activity was measured (C, D) as per the manufacturer's recommendations (Luciferase assay system with reporter lysis buffer, cat E4030; Promega Corporation). *p < .02. PMA, phorbol 12-myristate 13-acetate.

Article Snippet: Resting CD4 + T cells were isolated by negative selection from peripheral blood of healthy donors by the Rosettesep CD4 + cells isolation kit (STEMCELL Technologies, Inc.).

Techniques: Purification, Cell Culture, Positive Control, Western Blot, Infection, Virus, Luciferase, Activity Assay, Lysis