FIG 5 .
Mitochondrial changes in mutant epimastigotes. (A and B) Representative traces of oxygen uptake (A) and oxygen consumption rate (OCR) (B) by digitonin-permeabilized wild-type (WT), TcMCU-KO (MCU-KO), and TcMCUb-KO (MCUb-KO) epimastigotes. Additions were 100 μM ADP, 1 µg/ml oligomycin (Oligo), and 1.0 µM FCCP. (C to E) OCR after addition of ADP (respiration stimulated by oxidative phosphorylation [OXPHOS]), oligomycin (minimal respiratory rate), and FCCP (maximal respiratory capacity), respectively (n = 4), using the test system as in panels A and B. (F) Fluorescence microscopy of representative wild-type (WT), TcMCU-KO (MCU-KO), and TcMCUb-KO (MCUb-KO) epimastigotes labeled with MitoTracker Deep Red FM for estimation of mitochondrial mass. (G) Quantification of data in panel F. Relative fluorescence of MitoTracker Deep Red FM-labeled cells. Over 200 cells from three experiments with 20 random fields/experiment were analyzed (n = 3). (H) Relative citrate synthase activity of wild-type and mutant parasites, measured for enzymatic estimation of mitochondrial mass, considering Vmax values of WT to be 1.0 (n = 5). Values in panels C to E, G, and H are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.