Fig. 3

IBC altered mitochondrial morphologies and induced mitochondrial dysfunction of C. neoformans. (A) Mitochondrial morphologies of C. neoformans were assessed following incubation in YPD medium supplemented with various concentrations of IBC at 30 °C. MitoTracker Red CMXRos was used to stain diffuse, tubular, or fragmented morphologies, and mitochondria were then imaged using CLSM. The right images are the enlarged image of the white square on the left images. (B) MMP of C. neoformans was measured following incubation in YPD medium supplemented with various concentrations of IBC at 30 °C. Mitochondria were stained with JC-10 marker of MMP. The fluorescence microplate reader was used to quantify staining intensity. Experiment was repeated three times to ensure reproducibility. (C) Total intracellular ATP was measured for the C. neoformans following incubation in YPD medium supplemented with various concentrations of IBC at 30 °C. Normalized total cell lysates were determined for total intracellular ATP using a firefly luciferase bioluminescent assay. (D) Total intracellular ROS was measured for C. neoformans following incubation in the absence and presence of various concentrations of IBC. Cells were stained with DCFH-DA as a marker for intracellular ROS. The fluorescence microplate reader was used to quantify staining intensity. (E) IBC affects the electron transport chain function. Growth phenotypes of C. neoformans were assessed in the presence of electron transport chain inhibitors. Cells were normalized by OD₆₀₀ and subsequently cultured on YPD medium agar plates containing rotenone (0.5 mg/mL), salicylhydroxamic acid (SHAM) (2.5 mM), carboxin (50 µg/mL), antimycin A (3 µg/mL), and sodium azide (NaN₃) (0.5 mM) at 30 °C, respectively. Plates were imaged daily. Experiment was repeated two times to ensure reproducibility and representative images are shown