Fig. 1. Effective stepwise selection of Sb^III-resistant parasites.

Sb^III-sensitive or WT strain promastigotes were treated with rising Sb^III concentration for 6 months, selecting stepwise the resistant parasites. The middle point and the endpoint of drug-resistance selection were arbitrarily considered Sb^III moderately resistant (MR) and highly resistant (HR) strains, respectively. a The colorimetric MTT assay was used to estimate the EC50 value. Three conditions were evaluated per experiment: parasites growing without Sb^III (control), parasites growing under Sb^III pressure (treatment), and medium without parasites (blank). Negative (−) or positive (+) symbols reflect the absence/presence of parasites or drugs. The change from yellow (MTT) to purple (Formazan or reduced MTT) indicates cell viability. b EC50 values were calculated in three biological replicates with two technical replicates. Statistical analysis was performed by a one-way ANOVA analysis followed by Tukey’s multiple comparison test. Degrees of freedom for the numerator: 2. Degrees of freedom for the denominator: 6. F distribution value: 132.2. P value <0.001 (**), P value <0.0001 (****). P value <0.001 (**), P value <0.0001 (****). P value (WT vs. MR): 1.54 × 10⁻⁰³. P value (WT vs. HR): 8.38 × 10⁻⁰⁶. P value (MR vs. HR): 1.74E × 10⁻⁰⁴. Data are presented as mean and ±SD. The raw data of (b) are available in the Source Data.