Figure 6.

Brain endothelial cell from Trg-DN EGFR exhibit spontaneous targeting of intracellular T. gondii by LC3 and LAMP-1 as well as killing of the parasite dependent on autophagy. (a) Brain endothelial cells from Trg-Ctr and Trg-DN EGFR mice were infected with RH T. gondii. Monolayers were assessed by light microscopy. The percentages of infected cells, numbers of vacuoles containing T. gondii per 100 endothelial cells and tachyzoites per 100 endothelial cells were determined at 2 and 24 h. (b) Brain endothelial cells from Trg-Ctr and Trg-DN EGFR mice were infected with RH T. gondii. Lysates obtained at 0, 5 and 15 min post-challenge were subjected to immunoblot as indicated. (c,d) Brain endothelial cells from Trg-Ctr and Trg-DN EGFR mice were infected with RFP T. gondii (RH). Expression of LC3 was assessed by immunofluorescence 5 h after challenge (c). Insets represent magnification of areas around the parasites. Arrowheads indicate LC3 accumulation around the parasite (X630). Bar, 5 μm. (d) Expression of LAMP-1 was assessed by immunofluorescence 8 h after challenge. Arrowheads indicate LAMP-1 accumulation around the parasite. Bar, 5 μm. Bar graphs represent percentages of parasitophorous vacuoles surrounded by ring-like accumulation of LC3 or LAMP-1. (e) Endothelial cells were challenged with RH T. gondii followed by addition of lysosomal inhibitors (LI; leupeptin and pepstatin). Cells were examined as above. (f) Endothelial cells were transfected with control or ULK1 siRNA followed by challenge with RH T. gondii. Bars are mean ± SEM of 6 samples per group pooled from 2–3 experiments. **p < 0.01; *** p < 0.001 (Student’s t test).