Basal medium

The OCR and ECAR were measured using the Seahorse XF24 and XF96 instruments (Seahorse Bioscience, USA). Next, 2.5 × 10⁴ cells were plated in XF24 Cell Culture Microplates and 8000 cells in XF96. For the OCR test, the cell medium was changed by the assay medium (Seahorse Bioscience) supplemented with 2 mM glutamine, 10 mM glucose, and 1 mM pyruvate for 1 h in a 37 °C incubator prior to the measurements using the XF Cell Mito Stress Kit (Seahorse Bioscience). The concentrations of oligomycin and FCCP were 1.0 and 0.5 μM FCCP for SKOV3 and A549 cells, respectively. For the glycolytic metabolism test, the cells were incubated in the basal medium (Seahorse Bioscience) for 1 h in a 37 °C incubator prior to measurement using the Glycolytic Stress Test Kit (Seahorse Bioscience). The OCR and ECAR results were adjusted to the cell numbers. Finally, the cells that were prepared but not undergone any treatment were stained with CCK8 following spectrophotometric measurements at 450 nm (Molecular Device, USA).

Using Seahorse XF Extracellular Flux Analyzer XF24, the oxygen consumption rate (OCR) of cells was monitored in real-time (Agilent, Santa Clara, CA, USA). All tests were conducted according to the manufacturer’s guidelines. Briefly, 50,000 cells per well were coated with Cell-Tak (#Cat. 354,240, Corning, NY, USA) for 30 min on Seahorse XF24 Cell Culture Miniplate (Seahorse Biosciences, Agilent, Santa Clara, CA, USA). Prior to the assay, cells were equilibrated in a non-CO₂ incubator for 30 min with basal medium (Seahorse Biosciences, Agilent, Santa Clara, CA, USA) containing 1 mM pyruvate, 4 mM glutamine, and 1 mg/mL D-glucose. To establish a stable baseline OCR, the OCR under baseline measurements was measured at the beginning of the assay, prior to the injection of compounds. Following injection of stressed conditions (ATP-linked respiration by 1 µM oligomycin, spare respiratory capacity by 3 µM FCCP, proton leak by 0.5 µM rotenone, and 0.5 µM antimycin A), mitochondrial efficiency was evaluated. Using a Seahorse XFe24 Analyzer and the Seahorse XF Cell Mito Stress Test, the oxygen consumption rate (OCR) was determined (Agilent, Santa Clara, CA, USA). The experimental data were analyzed using the Wave2.6 control program (Agilent, Santa Clara, CA, USA).