Dako fluorescence mounting

U937 cells were seeded in Lab-Tek II Chamber Slides (Thermo Fisher Scientific, USA), and differentiated with PMA as previously described. Cells were dyed with 1 µg/mL Cell Tracker green (Thermo Fisher Scientific, USA) for 30 min, washed, and then incubated with inhibitors. T. cruzi trypomastigotes were dyed with 1 µg/mL Cell Tracker orange (Thermo Fisher Scientific, USA) for 30 min, washed, and incubated with the treated U937 with an MOI of 1. After 30 min of parasite incubation, cells were washed, and 24 hours later, cells were fixed with 4% PFA. Fixed cells were dyed with Hoechst (Thermo Fisher Scientific, USA) for 15 min and then mounted with Dako Fluorescence Mounting (Dako, USA). The cells were imaged using a Nikon Eclipse 400 epifluorescence microscope (Nikon, Japan). Parasite internalization was inferior to 5%, without significant differences among the different inhibitors, atorvastatin, and non-treated control (Supplementary Figure 3).

U937 cells were seeded in Lab-Tek II Chamber Slides (Thermo Fisher Scientific, USA), and differentiated with PMA as previously described. Cells were treated with inhibitors for 1 h, and then infected with T. cruzi for 30 min. After infection, cells were washed and fixed in 4% paraformaldehyde (PFA) for 15 min at RT and blocked with 5% bovine serum albumin in PBS containing 0.1% Triton X-100 for 2 h. Cells were then incubated with monoclonal anti-p65 antibodies (Cell Signaling, USA #8242) overnight at 4°C. The samples were washed with PBS and incubated with Alexa Fluor Ⓡ 555-conjugated anti-rabbit IgG Fab2 (Cell Signaling, USA #4413) for 1 h. Finally, nuclei were stained with DAPI for 5 min, and cells were mounted with Dako Fluorescence Mounting (Dako, USA). The cells were imaged using a Nikon Eclipse 400 epifluorescence microscope (Nikon, Japan), and images were analyzed by mean intensity using ImageJ software (ImageJ 1.47v).

2x10⁴ EA.hy926 cells/slide were seeded in Lab-Tek II Chamber Slide^TM (ThermoScientific, USA) and allowed to adhere overnight. Then, cells were incubated with 5 μM simvastatin or 20 μM benznidazole for 24 hours prior to T. cruzi- infection (Dm28c clone) at a MOI of 10. After 16 hours of infection, cells were fixed in 4% formaldehyde–0.1 M phosphate buffer (pH 7.3) for 10 min. Cells were blocked with 3% bovine serum albumin for 1 hour. Then, cells were incubated with monoclonal antibodies against E-selectin (1:100), VCAM-1 (1:250) and ICAM-1 (1:100) (from Abcam, UK) overnight at 4°C. The samples were washed with PBS and incubated with anti-rabbit IgG that had been conjugated with fluorescein (1:100) from Sigma-Aldrich for 1 h. Finally, nuclei were stained with DAPI for 5 minutes and mounted with Dako Fluorescence Mounting (Dako, USA). The cells were photographed using a Nikon Eclipse 400 fluorescence microscope (Nikon, Japan), and images were analyzed by mean intensity using ImageJ software (ImageJ 1.47v).