Figure 6

PPARγ RNA interference increases HIV replication in memory CD4⁺T cells. Memory CD4⁺ T-cells were isolated from thawed PBMC rested overnight by negative selection using magnetic beads. (A) Cells were stimulated via CD3/CD28 for 2 days, nucleofected with PPARγ or non-targeting (NT1) siRNA, and then cultured for an additional 24 h at 2x10⁶ cells/ml in the presence of IL-2. (B) RNA silencing efficiency was assessed by RT-PCR 24 h post-nucleofection (n = 6). (C-E) The effect of NT1 and PPARγ siRNA on cell viability and proliferation was assessed by flow cytometry at day 6 post-nucleofection (n = 6). Cell viability was monitored using the viability dye Vivid (C), cell counts were determined using FlowCounts fluorospheres (D), and cell proliferation was measured upon intracellular staining with Ki67 Abs (E). Cells were exposed to replication competent NL4.3BAL wt strain (10 ng HIV-p24/10⁶ cells) for 3 h at 37°C. Unbound virus was removed by extensive washing, and cells were maintained in culture in the presence of IL-2 up to 7 days post-infection. (F) Integrated HIV-DNA levels were quantified by real-time PCR in cells harvested at day 3 post-infection (n = 5). (G) Levels of HIV-p24 in cell supernatants were quantified by ELISA at days 3 and 6 post-infection (n = 6). (H) At day 7 post-infection, cells were harvested and the frequency of infected cells was analyzed by flow cytometry upon intracellular staining with HIV-p24 Abs. Shown is the frequency of HIV-p24⁺ cells (% and MFI, mean fluorescence intensity) upon nucleofection with PPARγ vs. NT1 siRNA in one representative subject (left panels) and statistical analysis in n = 5 different subjects (right panels). Paired t-Test values are indicated on the graphs.