*4.7. Detection of Intracellular ROS*

Intracellular ROS production was examined using MitoSox Red mitochondrial superoxide indicator according to the manufacturer's protocol and following our previously described method [52]. In brief, 24 h after plating the cells, the culture medium was replaced with fresh medium containing various concentrations of multifloroside (25-100 μM) or gefitinib (25 μM). After incubation for 48 h, cells were trypsinized, washed, and stained with MitoSox. Next, cells were washed, centrifuged to remove the supernatant, and resuspended in HBSS. Finally, ROS production in the cells was immediately monitored by flow cytometry (ACEA NovoCyte™, ACEA Biosciences, San Diego, CA, USA).

#### *4.8. Mitochondrial Membrane Potential (MMP) Assay*

MMP was analyzed using the JC-10 Mitochondrial Membrane Potential Assay Kit according to the manufacturer's instructions and following our previously described protocol [52]. In brief, 24 h after plating the cells, the culture medium was replaced with fresh medium containing various concentrations of multifloroside (25-100 μM) or gefitinib (25 μM). After incubation for 48 h, cells were trypsinized, washed, and stained with JC-10 dye. Next, cells were washed, centrifuged to remove the supernatant, and resuspended in PBS. Finally, the red/green fluorescence was detected by flow cytometry (ACEA NovoCyte™, ACEA Biosciences, San Diego, CA, USA).
