Figure 6

In vitro assays measuring activation and histone acetylation of the HIV-1 or SIV promoter by Pf extracts. (A-B) The effects of the synergistic activation of the HIV-1 promoter were determined by quantifying GFP-positive cells using flow cytometry 24 or 48 hours after treatment. (A) In total, 5 μg/ml Pf soluble extract induced HIV-1 promoter activation in J-Lat cells, leading to GFP expression. (B) Both PHA and VPA induced activation of the HIV-1 promoter in J-Lat cells and subsequent GFP expression. In contrast, PHA did not induce increased levels of histone acetylation in J-Lat cells, whereas VPA induced histone acetylation. (C) 5 μg/ml Plasmodium soluble extract increased histone acetylation levels in J-Lat cells, and 50 μg/ml Plasmodium soluble extract was sufficient to induce increased levels of histone acetylation in resting CD4+ T cells. This result was obtained from FACS assays. (D-E) Histone acetylation modifications in the HIV-1 LTR and SIV LTR promoters induced by Plasmodium soluble extract. Chromatin fragments from J-Lat cells or monkey PBMCs cultured for 24 hours with or without Plasmodium soluble extract or VPA were immunoprecipitated with anti-AcH3 or with normal rabbit serum (IgG) as a control. PCR was then used to amplify the DNA isolated from the immunoprecipitated chromatin. The relative dilution ratio of input/chip was 1.33. (D) Pf soluble extract induced an increase in the histone acetylation levels in the HIV-1 LTR. (E) Pf soluble extract induced an increase in the histone acetylation levels in the SIV LTR. The left panels of D and E show semi-quantitative results based on PCR products using input DNA, normal IgG control product or 24-hour CHIP product as the template. The data in this figure are presented as the means, and the error bars indicate the SD. One-way ANOVA was used to compare the variables in this figure.