ROS Measurement

Intracellular ROS levels were measured using a ROS assay kit (CA1410; Solarbio, Beijing, China). Overnight cultures of cmt1/2ΔΔ were diluted in fresh YPD medium to an optical density at 600 nm of 0.2. The cells were treated with 0.5 or 1mM Cu at 30 °C for 4 h (n=3). The cells were then collected and washed twice in phosphate-buffered saline (PBS). Next, about 2×10⁷ cells were stained with 20 μM 2′,7′-dichlorofluorescin diacetate (DCFH-DA) for 1 h. DCFH-DA (non-fluorescent) was hydrolyzed to dichlorofluorescin (DCFH) after entering cells. When DCFH is in the presence of ROS, it is oxidized to dichlorofluorescein (DCF), which is a strong green fluorescent substance. Fluorescence intensity was measured using microplate luminometers (Varioskan Flash; Thermo Fisher Scientific, Waltham, USA), with excitation at 488 nm and emission at 525 nm. The intracellular ROS level is shown as fluorescence value divided by the absorbance at 600 nm.