Fig 5. Effect of A6 on the cytosolic Ca²⁺, cytoplasmic ATP content and mitochondrial inner membrane potential of epimastigotes of T. cruzi at short-term and long-term.

The parasites were incubated or not (control) at different times with concentrations corresponding to 1.1, 2.2 or 4.4 μM A6 (as indicated in each corresponding figure). Quantification of cytosolic Ca²⁺ by fluorometry assay (λ_ex 490 nm and λ_em 518 nm): A) 0 h, B) 1h and C) 4 days. Quantification of cytoplasmic ATP levels assessed using a bioluminescent assay (λ 570 nm): D) 0 h, E) 1h and F) 4 days. Analysis of mitochondrial inner membrane potential (ΔΨm), the cells were labelled or not (unstained control) with 256 nM Rhodamine 123 (Rho123). In order to obtain a reference value for the parasites with the ΔΨm collapsed, parasites were incubated for 15 min with 10 μM FCCP before the measurements. Each histogram represents the distribution of fluorescence corresponding to ΔΨm for each treated population: G) 0 h, H) 1h and I) 4 days. The peaks of each population were used as a measurement of the average ΔΨm. Values of each bar represent the distances between each peak and that obtained by the reference (FCCP-treated parasites) in fluorescence intensity arbitrary units (A.U.) for each experimental condition: J) 0 h, K) 1h and L) 4 days. Results correspond to mean ± SEM of three independent experiments for each condition and were compared to the control using a t-test (**, P < 0.01; ****, P < 0.0001; ns, no significant).