Exposure to T. cruzi trypomastigotes or to exogenous sphingomyelinase induces formation of EEA1-positive endosomes in host cells. (A) After exposure to trypomastigotes for 20 min, HeLa cells were fixed and processed for quantification of intracellular parasites. The data correspond to the mean of triplicates ± SD. **, P = 0.0078, Student’s t test comparing control (white bar) and 10 µU/ml sphingomyelinase–exposed cells (black bar); ***, P < 0.0001, Student’s t test comparing control and 20 µU/ml sphingomyelinase–treated cells. (B) EEA1-positive endosomes in HeLa cells incubated (SMase) or not (control) with 20 µU/ml sphingomyelinase for 20 min. DAPI DNA stain (blue), anti-EEA1 antibodies (red). Bars = 20 µm. (C) EEA1-positive endosomes in HeLa cells incubated (T. cruzi) or not (control) with trypomastigotes for 20 min. DAPI, blue; anti-EEA1 antibodies, red. Bars, 20 µm. (D) After 20 min of infection, HeLa cells were incubated with anti–T. cruzi antibodies (green) to stain extracellular parasites, followed by permeabilization and staining with anti-EEA1 (red). Arrows point to EEA1 staining associated with the intracellular region of partially internalized parasites. DAPI (blue). Bars, 10 µm. (E) Quantification of the number of intracellular trypomastigotes in EEA1 positive vacuoles 20 min after invasion in the presence of sphingomyelinase. After infection, cells were incubated with anti–T. cruzi antibodies to stain extracellular parasites, followed by permeabilization and staining with anti-EEA1. The data correspond to the mean of triplicates ± SD. *, P < 0.04, Student’s t test; **, P = 0.002, Student’s t test, comparing control and treated cells. (F) After 20 min of infection, HeLa cells were fixed, permeabilized and stained with anti-EEA1 (red) and anti-Lamp1 (green) antibodies. DAPI, blue. Arrows indicate intracellular parasites within EEA1-enriched parasitophorous vacuoles and closely associated with Lamp1-containing lysosomes. Bars, 10 µm. These results are representative of three independent experiments.