Figure 6.

BSF cells with 90 to 95% reduced expression of Tb1 and OSCP have unchanged ΔΨm but are more sensitive to AAC and F_oF₁–ATPase inhibitors.A, flow cytometry analysis of TMRE-stained BSF Tb1 5’ RNAi (brick-red circles) and BSF OSCP RNAi (orange triangles) noninduced cells (−tet) and cells induced for 2 and 4 days (+tet, 2 days and 4 days) to detect changes in ΔΨm. The addition of FCCP served as a control for mitochondrial membrane depolarization (means ± SD, n = 4, Student’s unpaired t-test). B and C, mitochondrial membrane polarization detected using safranin O dye in digitonin-permeabilized BSF Tb1 5’ RNAi (B) and BSF OSCP RNAi (C) noninduced cells (−tet, black lines) and cells induced for 4 days (+tet, 4 days, red lines) in the presence of ATP. ATP, oligomycin (OLM), carboxyatractyloside (cATR), and SF 6847, an uncoupler, were added where indicated. cATR was added after OLM to test for any further depolarization of the mitochondrial membrane due to inhibition of the AAC, whose electrogenic activity can potentially contribute in the generation of ATP-stimulated ΔΨm. D and E, sensitivity of BSF Tb1 5’ RNAi– and BSF OSCP RNAi–noninduced cells (−tet, brick-red and orange full lines, respectively) and cells induced for 4 days (+tet, 4 days, brick-red and orange dashed lines, respectively) to carboxyatractyloside (cATR) (D) and to oligomycin (OLM) (E) estimated by resazurin cell-viability assay. The dose–response curves were calculated using GraphPad Prism 8.0 software. The calculated IC₅₀ values are shown beside the corresponding sample and are expressed in mM and in μg/ml for cATR and OLM, respectively. ΔΨm, mitochondrial membrane potential; AAC, ADP/ATP carrier; BSF, bloodstream form; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; OSCP, oligomycin sensitivity-conferring protein; TMRE, tetramethylrhodamine ethyl ester.