Figure 3

Extracellular and intracellular cytolytic activity of AnlO. (A) Extracellular cytolytic activity of AnlO. THP-1 cells or primary human macrophages were treated with different concentrations of purified AnlO in serum-free medium for one hour at 37°C. Cytolytic activity of AnlO was assayed by measuring the relative activity of lactate dehydrogenase (LDH) released into the culture following cell lysis. (B) Inhibition of AnlO activity by human plasma. AnlO was serially diluted and incubated with human plasma (heat inactivated) for 20 minutes on ice and then added to THP-1 cells in serumfree medium for one hour at 37°C. Cytolytic activity of AnlO was assayed by measuring the relative activity of lactate dehydrogenase (LDH) released into the culture. In the controls, AnlO was directly added into THP-1 cells without being neutralized by plasma. (C) Intracellular cytolytic activity of AnlO. pNL-GFP-RRE-SA (middle panels, GFP) or pNL-AnlO-GFP-RRE-SA (right panels, AnlO-GFP) was co-transfected with pCMVΔ8.2 into HEK293T cells. At day 2 to 4 after cotransfection, cells were analyzed by flow cytometry for GFP expression. PI is propidium iodide. Mock transfected cells (left panels, Cell) were used as controls for the GFP and PI positive population.