*2.4. Effect of Pro-Oxidant and Antioxidant Molecules on Intracellular Amastigotes*

To analyze the effect of oxidative burst on the infection, macrophages were collected from the peritoneal cavity of uninfected male BALB/c mice (5–6 weeks) after the injection of 10 mL of RPMI medium (LGC Biotecnologia, Cotia, Brazil). Peritoneal macrophages were resuspended in RPMI supplemented with 10% FBS and plated in 24-well plates (3 × <sup>10</sup><sup>5</sup> cells/well) for 24 h at 37 ◦C and 5% CO2. After that, culture medium was replaced, and cells were infected or not with promastigotes (10:1 parasites/host cell) of each strain. After 4 h of incubation, cultures were washed to remove non-internalized parasites and maintained under the same conditions until complete 24 h of infection. Infected macrophages were treated or not with 150 μM hydrogen peroxide (H2O2), 2 mM NaNO2, 30 U/mL superoxide dismutase from bovine erythrocytes (SOD; Sigma-Aldrich, St. Louis, MO, USA), 40 U/mL catalase from bovine liver (Sigma-Aldrich, St. Louis, MO, USA), 1 μM mitoTEMPO (Santa Cruz Biotechnology, Dallas, TX, USA), or 2 mM Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME; Sigma-Aldrich, St. Louis, MO, USA), for 48 h. The culture was then stained with fast panoptic (Laborclin, Pinhais, Brazil), and percentage of infected cells, number of protozoa per cells, and infection index (percentage of infected host cells multiplied by the number of parasites per cell) were calculated.

As host toxicity control, non-infected macrophages (5 × 104 cells/well) were also treated with the compounds for 48 h at 37 ◦C and 5% CO2. After the treatment, 10 μL PrestoBlue was added to the final concentration of 10% and cells were incubated for 2 h at 37 ◦C and 5% CO2. The measurements were performed at 560 and 590 nm in a SpectraMax M3 fluorimeter.

#### *2.5. ROS and RNS Release by Infected Macrophages*

To evaluate the levels of reactive species released from infected cells, peritoneal macrophages were obtained as described above and incubated with LPS (100 ng/mL) plus IFN-γ (10 ng/mL) for 30 min. Cells were then infected for 72 h, and culture supernatants were collected for RNS detection by Griess reagent (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer's instructions. Briefly, 100 μL of culture supernatant was added to the same volume of colorimetric reagent and incubated at room temperature for 30 min, and absorbance was read at 540 nm. Values were compared against an NaNO2 standard curve, ranging between 50 and 3 μM. In parallel, macrophages were resuspended in 100 μL respiration buffer consisting of 65 mM KCl, 10 mM Tris-HCl (pH 7.2), 1 mM MgCl2, and 2.5 mM potassium phosphate monobasic [26]. After that, 100 μM Amplex Red reagent (Invitrogen, Carlsbad, CA, USA) and 50 U/mL horseradish peroxidase (HRP; Sigma-Aldrich, St. Louis, MO, USA) were added and the cells were incubated for 30 min at 37 ◦C. The supernatants were then collected and analyzed to ROS presence by a SpectraMax M3 fluorimeter.

#### *2.6. Protein Extraction and Sample Preparation*

For proteomic analysis, promastigotes were treated or not with 1/5 IC50/4 h NaNO2 (for each strain) in HBSS for 4 h at 25 ◦C. Then, parasites were washed three times with PBS and resuspended in lysis buffer for proteomics sample preparation. Because we are interested in the early effects of NO on proteome remodeling, we choose a sublethal dose to challenge the parasites. Additionally, with this way we avoid results related to parasites' death. These assays were performed in quadruplicate (four independent biological replicates), and samples were prepared accordingly to previous reports [25]. Briefly, parasites were lysed in a buffer containing 0.05 M Tris-HCl (pH 7.6), 0.05 M DTT and 2% SDS (*w*/*v*) and boiled in a water bath for 5 min. After chilling to room temperature, the SDS lysates were clarified by centrifugation at 10,000×g for 5 min and processed in 30k filtration units (Millipore, Burlington, MA, USA) using the MED-FASP (Multi-Enzyme Digestion—Filter Aided Sample Preparation) protocol. Proteins were digested sequentially with endoproteinase LysC and trypsin in a 1/100 enzyme to protein ratio [27,28]. Peptides were collected, concentrated, and desalted on a C18 reversed phase column.
