Figure 5

(A–C) pp242 (A), Torin1 (B) and rapamycin (C) suppress Tat-dependent HIV LTR activation after 24h in a dose-dependent manner in luciferase assays with the HIV LTR construct in Jurkat cells. Tat was used at increasing doses (0, 0.05, 0.25 and 1.25 ng). Data are represented as mean +/− SD of triplicate values of relative luciferase units (RLU) normalized by protein content (representative of at least two independent experiments).

(D) pp242, Torin1 and rapamycin treatment differentially affects Tat-dependent HIV LTR activation in the context of integrated provirus (TZM-bl cells). Tat was used at increasing doses (0, 1, 10 and 100 ng plasmid). TZM-bl cells were transfected with indicated amounts of Tat plasmid and treated with DMSO, Torin 1 (100, 200nM), pp242 (0.5, 1µM), or Rapamycin (10, 50nM). 24h post-treatment, cells were lysed and HIV transcription was measured via luciferase activity. Data are represented as mean +/− SEM of fold change of RLU normalized by protein content.

(E) pp242, Torin1 and Rapamycin reduce basal LTR activity in A72 Jurkat cells (integrated LTR-GFP) measured by percentage of GFP-positive cells.

(F–H) Primary CD4 T cells were isolated from a donor’s blood and either pre-treated with pp242 (0, 8, 40, 200 and 1000 nM) for 30 minutes (F), left untreated (G), or pre-treated with Okadaic acid (0, 10, 100 or 1000 nM) for 30 minutes (H). The cells were co-stimulated with CD3/CD28 antibodies for 2 h. Cells were harvested for protein extraction right before (0 min) and after (120 minutes) adding the CD3/CD28 beads. Proteins were extracted as explained in Experimental Procedures. Whole cell protein extracts from (G) were either phosphatase-treated or not. Extracts were run on an SDS-PAGE gel and immunoblotted for the indicated proteins (CDK9, phospho-S6 (Ser240/244) and S6).

See also Figure S3