transduction, the transduced cells were treated with lipopolysaccha- ride (LPS, 50 mg/ml) and cycloheximide (CHX, 10 mg/ml), which impose stress on the cells, in the presence or absence of three different ALP compounds, edelfosine, perifosine or miltefosine (see Figure 1 for their chemical structures), for 24 hours. Lysates were then prepared and applied to western blots for examination of both total Akt and serine-473 phospho-specific Akt levels. Ratios between phospho-specific Akt and total Akt were determined as marked in Figure 2A. Note that since treatment with LPS/CHX alone induced a significant amount of death of the untransduced/control cells within 24 hours as previously reported [19], the analysis of the untransduced cells in this experiment was omitted. As shown in Figure 2A, the DHIV-GFP vector-transduced cells exhibit a low level of Akt phosphorylation in the absence of LPS/ CHX treatment (‘‘C’’), while LPS/CHX treatment (‘‘L’’) signif- icantly induced the phosphorylation of Akt in the vector- transduced CHME5 cells. However, upon treatment with both LPS/CHX and ALP compounds, the phosphorylation of Akt in transduced cells decreased, suggesting that the ALP compounds effectively block the LPS/CHX stress-induced Akt phosphoryla- tion in the HIV-1 vector-transduced CHME5 cells. We previously reported that the expression of Tat is responsible for the cytoprotective effect of HIV-1 expressing macrophages and CHME5 cells [5,19], which is supported by our previous observation that Tat expression elevates the phosphorylation of Akt and GSK3b in CHME5 (Figure S2). Thus, we tested if the ALP compounds also block the stress-induced Akt phosphorylation of the CHME5 sublines that were previously constructed by stably transfecting with pCDNA3.1-hygro [(2) Tat] or pTat101 [(+) Tat] expressing the 101 amino acid M-tropic YU-2 Tat protein [19]. In this test, an ELISA-based phospho-Akt assay was employed to Figure 1. Structures of alkylphospholipid compounds used in this study. doi:10.1371/journal.pone.0013121.g001 Targeted HIV-1 Reservoirs determine the level of Akt phosphorylation. The lysates of (+) Tat CHME5 subline cells were prepared in the presence of LPS/CHX alone or both LPS/CHX and an ALP compound. Lysates, normalized by protein level, were applied for the ELISA-based phospho-Akt assay. Again, note that the control (2) Tat cells were not analyzed in this experiment because the LPS/CHX treatment very effectively induces the death of the control cells as reported previously [19] and shown later in this study. As shown in Figure 2B, the LPS/CHX treatment significantly elevated the Akt phosphorylation in the (+) Tat subline cells, supporting that stress- induced cell survival activation in the Tat expressing CHME5. However, the treatment with the ALP compounds alone did not affect the Akt phosphorylation. It is also important to note that HIV-1 vector transduction alone does not increase the Akt phosphorylation in the absence of the LPS/CHX (Figure 2). However, as shown in Figure 2B, indeed, the treatment with the ALP compounds reduced the LPS/CHX induced Akt phosphor- ylation in the Tat expressing cells, suggesting the ALP compounds effectively counteract the stress-induced Akt phosphorylation. Effect of ALP compounds on kinase activity of Akt in CHME5 cells Phosphorylation of Akt activates its kinase activity, allowing the enzyme to phosphorylate a number of downstream effectors such as GSK3b [20]. We investigated if the ALP compounds, which reduced the phosphorylation of Akt kinase, also block the kinase activity of Akt. For this, the phosphorylation of GSK3b, a downstream substrate of Akt kinase was monitored. The same CHME5 cell lysates used for examining the phosphorylation of Akt kinase (Figure 3) were analyzed for determining both total and phosphorylated GSK3b. As shown in Figure 3A, the LPS/CHX stress elevated the phosphorylation of GSK3b in the DHIV-GFP vector transduced, compared to the untreated cells. However, the treatments with perifosine, edelfosine or miltefosine reduced the phosphorylation of GSK3b even in the presence of LPS/CHX, compared to the LPS/CHX alone treated cells. In addition, as shown in Figure 3B, the three ALP compounds reduced GSK3b phosphorylation to the control cell level in the (+) Tat CHME5 subline cells even in the presence of the LPS/CHX stress. The data shown in Figure 3 support that perifosine, edelfosine, and miltefosine block the kinase activity of Akt in both HIV-1- and Tat-expressing cells. These data mechanistically postulate that the three ALP compounds tested may be able to abolish the cytoprotective phenotype of HIV-1 and Tat-expressing CHME5 cells. Counteracting cytoprotective phenotype of CHME5 expressing HIV-1 proteins or Tat by ALP compounds Next, since the data presented in Figures 2 and 3 demonstrated that the ALP compounds inhibit both stress-induced Akt activation and Akt kinase activity, we tested if the ALP compounds counteract the elevated survival capability of the CHME5 cells expressing HIV-1. First, CHME5 cells were transduced by DHIV- GFP (D3) and cultured for 48 hours, yielding .90% transduction as determined by FACS for the GFP expression. Cells were then exposed to LPS and CHX, which impose stress on the cells, for 24 hours in the presence and absence of the ALP compounds (edelfosine and miltefosine, Figure 4A). Cells were stained with ethidium homodimer for visualization of dead cells; the amount of dead cells (red) among transduced cells (green) was determined. As shown in Figure 4A, the LPS/CHX treatment induced cell death (,20%) in the cells alone, and induced little death among transduced cells (,2%), supporting the cytoprotective effect of PLoS ONE | www.plosone.org 3 September 2010 | Volume 5 | Issue 9 | e13121