Increasing the Fungicidal Action of Amphotericin B by Inhibiting the Nitric Oxide-Dependent Tolerance Pathway
L-NAME increased and accelerated AmB-induced superoxide radical accumulation, membrane permeabilization, and intracellular superoxide radical levels. (a-b) Accumulation of superoxide radicals (a) and membrane permeabilization (b) in S. cerevisiae cells treated either with 0 μM (black), 5 μM (orange), or 10 μM (blue) AmB in the presence (dashed lines) or absence (solid lines) of 200 mM L-NAME during 3 h in 15 min intervals. Log-rank tests were performed to analyse significant differences between AmB treatment and treatment of AmB in combination with 200 mM L-NAME for each AmB dose. Data of at least 3 independent biological experiments is presented (). and represent and , respectively. (c-d) Intracellular DHE fluorescence in S. cerevisiae cells treated with 10 μM AmB in the absence (c) or presence (d) of 200 mM L-NAME. Single cells were monitored for their DHE fluorescence during treatment for 3 h in 15 min intervals using fluorescence microscopy and the DMF platform. The fluorescence intensity of each cell is presented as arbitrary units (AU), and each dot represents a single cell. Means and standard errors of the means (SEMs) of at least 3 independent biological experiments (), with at least 780 cells each, are presented. Two-way ANOVA followed by Tukey multiple comparison test was performed to analyse significant differences in DHE fluorescence intensity. ,, and represent ,, and , respectively. (e) DHE fluorescence intensity of individual cells over time. A selection of 35 cells was randomly chosen and is representative for more than 3000 cells that were analysed in this study. Each plot represents the DHE fluorescence intensity, measured every 15 min, of one representative cell over the whole duration of the experiment, that is, 180 min.
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