Ceramide-enriched T. cruzi-containing vacuoles are dependent on ASM activity. HeLa cells treated with control or ASM siRNA were exposed to trypomastigotes for 20 min, fixed, and processed as described in Fig. 7. Confocal Z series analysis performed in 15 fields for each condition revealed a smaller percentage of ceramide positive vacuoles in cells treated with ASM siRNA (24%) when compared with control siRNA (60%). Representative images of single optical planes for each condition are shown (two examples of control siRNA and two examples of ASM-siRNA). Ceramide, red; Lamp1, green; DAPI, blue. The arrows indicate parasites within vacuoles positive for ceramide staining in cells treated with the control siRNA. Arrowheads point to the DAPI-stained kinetoplasts of trypomastigotes within vacuoles negative for ceramide staining, in cells treated with ASM siRNA. Bars, 5 µm. These results are representative of three independent experiments.