2.6. Measurement of ROS and MDA Levels in Yeast upon Treatment with GENI

ROS was measured as described in our previous study [20]. In brief, the BY4741 yeast strain in glucose liquid medium treated with 0, 1, and 3 µM GENI or 10 µM RES was cultured with shaking at 28 °C and 160 rpm for 12 h. Each group was washed with PBS, added with 2′,7′-dichloro-dihydrofluorescein diacetate fluorescent probes to a final concentration at 10 µM, incubated for 1 h with shaking in the dark, and washed with PBS. The DCF fluorescence was determined using a microplate reader (BioTek, VT, USA) at 488 nm excitation and 525 nm emission wavelengths.

The MDA was measured as described in our previous study [20]. In brief, the BY4741 yeast strain in glucose liquid medium treated with 0, 1, and 3 µM GENI or 10 µM RES was cultured with shaking at 28 °C and 160 rpm for 12 h, washed with PBS, sonicated on ice for 5 min for protein extraction, and centrifuged at 4 °C and 12,000 rpm for 10 min to obtain the supernatant as protein samples. The protein concentration was determined using the BCA kit (CoWin Biotech, Beijing, China). According to the MDA kit (Nanjing Jiancheng Bioengineering, Nanjing, China) method, the MDA content of each group was determined. Detailed steps are shown in the Supporting Information (S2.1).

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