FIG 2.

PIM-1 inhibitor IV prevents activation induced HIV-1 reactivation. (A) Latently HIV-1-infected CA5 reporter T cells were stimulated with the phorbol ester PMA (3 ng/ml) in the presence or absence of PIMi IV (10 μM), and reactivation was measured as the percentage of GFP-positive cells by using flow cytometric analysis. (B) PIMi IV was titrated on CA5 T cells against TNF-α (10 ng/ml) or PMA (3 ng/ml) as HIV-1-reactivating agent. The level of HIV-1 reactivation was determined as the percentage of GFP-positive cells using flow cytometric analysis and plotted over the PIMi IV concentration. CA5 T cells were preincubated for 6 h with PIMi IV prior to triggering HIV-1 reactivation. rel, relative. (C) PIMi II was titrated on CA5 T cells against TNF-α (10 ng/ml) or PMA (3 ng/ml) as HIV-1-reactivating agent. The level of HIV-1 reactivation was determined as the percentage of GFP-positive cells using flow cytometric analysis and plotted over the PIMi II concentration. CA5 T cells were preincubated for 18 h with PIMi II prior to triggering reactivation. (D) PIMi IV was titrated on chronically actively HIV-1-infected JNLG T cells. GFP mean channel fluorescence (GFP-MCF) was determined as a quantitative surrogate marker of HIV-1 expression. (E) The latently HIV-1-infected T cell lines CA5 and EF7 were retrovirally transduced to overexpress PIM-1 protein. Following retroviral transduction, bryostatin, an anticancer drug candidate that triggers PKC/NF-κB activation, was titrated on CA5-PIM or EF7-PIM cells (PIM) and the level of HIV-1 reactivation as measured by GFP expression was compared to that of the parental cells (control). (F) PIM-1 expression in CA5 T cells was knocked down using two different anti-PIM-1 shRNA constructs (shPIM#10 and shPIM#22), and PIM-1 shRNA-transduced clones were generated. For an unbiased, representative cross-section of CA5-shPIM#22 cell clones, TNF-α was then titrated on either control CA5 T cells (black symbols), a population of CA5 T cells that were transduced with a scrambled shRNA and then puromycin selected (large gray triangles), and the various generated PIM-1 shRNA transduced clones (gray symbols/lines; all left panel) and determined as the percentage of GFP-positive cells as a surrogate marker of HIV-1 reactivation. The effect of PIM-1 knockdown on concentration-dependent TNF-α-mediated HIV-1 reactivation was detailed for four CA5-shPIM#10 cell clones (middle panel), and achievable HIV-1 reactivation levels (percentage of GFP-positive cells) were correlated with PIM-1 expression as determined by Western blotting for PIM-1 (right panel). The numbers over the inset showing the Western blot data indicated the band intensities (in arbitrary units) for PIM-1 expression.