Fig. 7.

Mitochondrial membrane potential. Parasites were exposed to 0.5 mM H₂O₂, harvested every hour, incubated with the TMRM dye and analysed by flow cytometry to detect the loss of mitochondrial membrane potential.

A. Mitochondrial membrane potential and cell integrity analysis after 3 h H₂O₂ treatment of parasites expressing wild type (WT), GFP, LmjMCA–GFP, cd–GFP, LmjMCA-Flag and LmjMCA-Flag H147A/C202A. Negative (untreated) and positive (10 mM H₂O₂, 1 h) controls are shown in the two first dot plots respectively. Percentages of healthy (TMRM-positive, Live/Dead-negative) and necrotic (TMRM-negative, Live/Dead-positive) cells are shown in bold.

B. Mitochondrial membrane potential analysis over 6 h H₂O₂ treatment of parasites expressing WT, GFP, GFP-tagged full-length metacaspase (LmjMCA–GFP) and metacaspase catalytic domain (cd–GFP).

C. Mitochondrial membrane potential analysis over 6 h H₂O₂ treatment of parasites expressing WT, GFP, Flag-tagged full-length metacaspase (LmjMCA-Flag) and its catalytically inactive version (LmjMCA-Flag H147A/C202A).

Lower panels show immunoblots of protein extracts from each analysed sample with the level of expression of LmjMCA–GFP, cd–GFP and LmjMCA-Flag, LmjMCA-Flag H147A/C202A using anti-GFP and anti-Flag antibodies respectively.