*2.9. Analysis of 2-NBDG Uptake*

Promastigotes (5 × <sup>10</sup><sup>6</sup> protozoa/mL) were treated with 1/5 IC50/4 h NaNO2 in HBSS at 25 ◦C for 4 h. After that, protozoa were washed with PBS and incubated with 300 μM 2-(N- (7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) (2-NBDG, Molecular Probes, Eugene, OR, USA), a fluorescent glucose analogue, for 30 min. The 2-NBDG specificity was monitored by the parasite incubation at 4 ◦C for the same amount of time, to decrease the compound uptake. 2-NBDG+ parasites were analyzed using a CytekDxP multi-color upgrade flow cytometer (Cytek, Fremont, CA, USA). A total of 10,000 events were acquired in the region previously established by protozoa morphology, and analyses were performed in Summit 6.1 software (Beckman Coulter, Brea, CA, USA).

#### *2.10. Analysis of Oxygen Uptake*

Promastigotes (5 × 106 protozoa/mL) were treated with 1/5 IC50/4 h NaNO2 in HBSS at 25 ◦C for 4 h. After that, protozoa were washed with PBS and resuspended (2.5 × 107 protozoa/mL) in a respiration buffer at 25 ◦C with continuous stirring in a highresolution Oxygraph-2K (Oroboros Instruments, Innsbruck, Austria) [26]. Mitochondrial respiration was confirmed by the addition of 2 μM antimycin A (AA, Sigma-Aldrich, St. Louis, MO, USA) to obtain the residual O2 consumption (ROX). O2 concentration and flux data were acquired using DatLab software (Oroboros Instruments, Innsbruck, Austria).
