Metabolic profiling

The key parameters of glycolytic function including basal glycolysis, glycolytic capacity, and glycolytic reserve, were assessed using a Seahorse XF glycolysis assay (Agilent Technologies, Berlin, Germany) according to the manufacturer’s instructions. Prior to the assay, XF sensor cartridges were hydrated. To each well of an XF utility plate, 1 mL of Seahorse Bioscience calibrant was added and the XF sensor cartridges were placed on top of the utility plate, and kept at 37 °C incubator without CO₂ for 12 hrs. MCF7 or MDA-MB-231 cells were cultured in XF24-well cell culture microplates (Agilent) at a density of 4 × 10⁴ cells/per well and then incubated for 24 h at 37 °C under 5% CO₂ atmosphere. The cells were incubated with iodoacetate (5, 10, 15, 20 µM) for instantaneous, 4 h or 12 h pre-incubation. Oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were measured simultaneously for 16 min to establish a baseline measurement²⁴. Glycolysis, glycolytic capacity, and glycolytic reserve were calculated by subtracting the average rates before and after the addition of glucose (10 mM), ATP synthase inhibitor oligomycin (1.0 µM) and 2-deoxy-D-glucose (2-DG) (50 μM). These three compounds were injected consecutively with a specific time gap and ECAR values were measured after each injection.

Different parameters of respiration, basal respiration, coupling efficiency, and spare respiratory capacity, were investigated by using a Seahorse XF-24 cell Mito Stress assay according to the manufacturer’s instructions. The following electron transport inhibitors were added; oligomycin (1.0 μM), FCCP (0.5 μM) and a mixture of antimycin-A (0.5 μM) and rotenone (0.5 μM). The different parameters of respiration were calculated by subtracting the average respiration rates before and after the addition of the electron transport inhibitors The calculated parameters included: basal respiration (baseline respiration minus antimycin-A post injection respiration), ATP turnover (baseline respiration minus oligomycin post injection respiration), maximal respiratory capacity (FCCP stimulated respiration minus antimycin-A post injection respiration) and reserve respiratory capacity (FCCP stimulated respiration minus baseline respiration)²⁵.