Mitochondrial staining

Mitochondrial activity was assessed with MitoTracker Orange (#M7510, Invitrogen), whose accumulation in mitochondria is dependent upon membrane potential. Mitochondrial mass was determined using MitoTracker Deep-Red (#{"type":"entrez-nucleotide","attrs":{"text":"M22426","term_id":"197107","term_text":"M22426"}}M22426, Invitrogen), localizing to mitochondria regardless of mitochondrial membrane potential [39,40]. MCF7 transfected cells were seeded for 48 hours. MCF7-EV control cells were processed in parallel. After 48 hours, cells were incubated with pre-warmed MitoTracker staining solution (diluted in PBS/CM to a final concentration of 10 nM) for 30-60 minutes at 37°C. All subsequent steps were performed in the dark. Cells were washed in PBS, harvested, and re-suspended in 300 μL of PBS/CM. Cells were then analyzed by flow cytometry. Data analysis was performed using FlowJo software.