Figure 1.

The effect of AdOx on HIV-1 production in CEM T-cells and HEK293T cells. A) CEM cell confluence and viability was assessed using a trypan blue exclusion assay and counting with a hemocytometer. The cell confluence and viability measured in two independent assays and the standard deviation of the mean are shown. B) Western blot of cell lysates from transfected HEK293T cells with the anti-asymmetric dimethylarginine antibody ASYM24 demonstrating a clear reduction in amounts of methylated proteins present in cells treated with 10 μM AdOx compared with cells not treated with AdOx. C) Culture supernatant from the treated CEM cells were assayed for CAp24 and RT content. Shown are representative results from two experiments with the standard deviations of the mean shown. HEK293T cells were transfected with the proviral plasmid pNL4.3 and treated with AdOx as described in the Materials and Methods. Culture supernatant from treated HEK293T cells were assayed for CAp24 and RT content 48 h post transfection. Shown are representative results from two experiments with the standard deviation of the mean shown. D) Whole cell lysates were prepared from infected CEM or transfected HEK293 cells. CAp24 was measured by ELISA of serially diluted lysates and total protein concentration was determined by Bradford assay. Shown is the CAp24 concentration/mg total protein. These experiments were performed from two to four times and the standard deviation of the mean is shown. E) HIV-1 produced by control and AdOx-treated CEM cells were partially purified by ultracentrifugation through a 20% sucrose cushion. The viral pellet was resuspended in RT lysis buffer and the RT and CAp24 levels were measured. The concentration of the initial filtered supernatant, and the amount of residual CAp24 and RT were also measured so that the % recovery could be determined. This experiment was performed twice and a representative result is shown. F) The ratio of the absolute values measured in (E) are shown.