Figure 5. TEM and flow cytometry assays after treatment with SBIs at the EC₁₀₀/24 h.

(A) TEM images after 18 hours of exposure to SBIs at the EC₁₀₀/24 h, showing marked cell degradation, with kinetoplast disruption (* in (i)) and cell lysis (ii); the occurrence of reservosome lysis is highlighted in (iii) and (iv). These morphological patterns are similar to those observed during cell death by necrosis. TEM results were similar for the two SBIs and the drug name is not shown. Scale bars: (i) and (ii), 1 µm; (iii) and (iv), 0.5 µm. (B) Flow cytometry analysis of T. cruzi necrotic death in reponse to SBIs at the EC₁₀₀/24 h. (i) Analysis of relative intracellular calcium concentrations by fluo-4-AM staining, after 0.5 to 12 hours. A rapid increase in fluo-4-AM fluorescence with respect to control cells can be seen after exposure to the two SBIs at the EC₁₀₀/24 h. (ii) Assay of mitochondrial membrane depolarization by R123 staining; time-dependent mitochondrial depolarization can clearly be seen by comparison with control cells. (iii) Cell viability analysis based on propidium iodide staining; the percentage dead cells (PI-positive) is plotted as a function of drug exposure time. Note the differences in cell lysis kinetics for the two drugs: the experimental points were optimally adjusted by a sigmoidal curve for lovastatin and by a negative exponential curve for ketoconazole. The raw flow cytometry plots can be seen in Figure S4.