Figure 2.

Analysis of parameters of cell viability, integrity, and intercellular ROS levels during menadione treatment. Kinetic analysis (200 min) of intracellular ROS levels (A), cell death (B), plasma membrane integrity (C), and mitochondrial function/integrity (D) in wild-type and btn1-Δ cells exposed to 0.1 mM menadione, and visualization by epifluorescence microscopy of loss of membrane integrity (shown in red) and intracellular ROS (shown in green) in wild-type and btn1-Δ cells after 30-min treatment with 0.1 mM menadione (E). (A) Percentage of cells with increased ROS levels, assessed by flow cytometry using the fluorescent probe MitoTracker Red CM-H2XRos at 50 μg/ml. (B) Percentage of viable cells determined by the counting of colony-forming units. (C) Percentage of cells with impaired cytoplasmic membrane, assessed by flow cytometric quantification of cells labeled with propidium iodide at 5 μg/ml. (D) Percentage of cells with impaired mitochondrial function or integrity, assessed by flow cytometry using the fluorescent probe rhodamine 123 at 25 μg/ml. *p ≤ 0.001 versus wild-type, t test; n = 5. (E) Loss of membrane integrity and intracellular ROS levels were visualized by colabeling cells with dihydrorhodamine 123 at 15 μg/ml (green fluorescence) and propidium iodide at 5 μg/ml (red fluorescence).