Figure 4: Menadione-generated mitochondrial ROS induces differentiation.

Mid-log phase WT promastigots were resuspended at a concentration of 5×10⁶ cells/mL in regular medium and treated with DMSO (0), 1, 3, 5, 7.5 and 10 μM menadione respectively for 24 h. (A) Cells were stained with FDA and live cells were counted with a hemocytometer. Starting cell concentration (5×10⁶ cells/mL) is indicated by the broken line. A one-way ANOVA with Dunnett’s post-hoc test was performed, with 24 h untreated values as the control. (B) Live cells with and without a flagellum were enumerated. A one-way ANOVA with Dunnett’s post-hoc test was performed, with 24hr untreated as the control column. (A-B) Our results represent the mean of three independent biological replicates (n=3). Error bars represent standard error of measurement. P values are indicated in the figure. (C-D) Representative images showing the morphology (DIC) and viability (FDA fluorescence) of parasites treated with DMSO or with increasing concentrations of menadione. Bars = 14μm. Viable amastigote-like parasites are indicated by arrows.