The bioenergetic function of MCF-7 and MCF-10A cells in response to Mito-CP or 2-DG was determined using a Seahorse Bioscience XF24 Extracellular Flux Analyzer (Seahorse Bioscience). MCF-7 or MCF-10A cells were seeded in specialized V7 Seahorse tissue culture plates. One hour prior to the start of the experiment, cells were washed and changed to unbuffered assay medium adjusted to pH 7.4, final volume 675 µl (MEM-α for MCF-7, DMEM/F12 for MCF-10A). After establishing the baseline oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), Mito-CP (1 µM) or 2-DG (5 mM) were administered through an automated pneumatic injection port of XF24. The changes in OCR and ECAR were monitored for 4 h. The resulting effects on OCR and ECAR are shown as a percentage of the baseline measurement for each treatment.
To determine the mitochondrial and glycolytic function of MCF-7 and MCF-10A cells in response to Mito-CP, Mito-Q, and 2-DG, we used the bioenergetic function assay previously described with several modifications (31 (link),32 (link)). After seeding and treatment as indicated, MCF-7 cells and MCF-10A cells were washed with complete media and either assayed immediately, or returned to a 37°C incubator for 36 or 60 h. The cells were then washed with unbuffered media as described above. Five baseline OCR and ECAR measurements were then taken before injection of oligomycin (1 µg/ml) to inhibit ATP synthase, FCCP (1–3 µM) to uncouple the mitochondria and yield maximal OCR, and antimycin A (10 µM) to prevent mitochondrial oxygen consumption through inhibition of Complex III. From these measurements, indices of mitochondrial function were determined as previously described (31 (link),32 (link)).
To determine the mitochondrial and glycolytic function of MCF-7 and MCF-10A cells in response to Mito-CP, Mito-Q, and 2-DG, we used the bioenergetic function assay previously described with several modifications (31 (link),32 (link)). After seeding and treatment as indicated, MCF-7 cells and MCF-10A cells were washed with complete media and either assayed immediately, or returned to a 37°C incubator for 36 or 60 h. The cells were then washed with unbuffered media as described above. Five baseline OCR and ECAR measurements were then taken before injection of oligomycin (1 µg/ml) to inhibit ATP synthase, FCCP (1–3 µM) to uncouple the mitochondria and yield maximal OCR, and antimycin A (10 µM) to prevent mitochondrial oxygen consumption through inhibition of Complex III. From these measurements, indices of mitochondrial function were determined as previously described (31 (link),32 (link)).