Wave2.6 control program

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).

The HEK293T cells (3.75 × 10⁴ cells), H1299 (1.75 × 10⁴ cells) and MCF-7 (2.8 × 10⁴ cells) were seeded and incubated in Agilent Seahorse XF24 cell culture microplates for 24 h 37 °C with 5% CO₂. HEK293T cells were then exposed to 0, 25, and 50 µM EA and MDSA for 4 h, while H1299 and MCF-7 cells were exposed to 0, 75, and 150 µM EA and MDSA for 3 h. After 30 min of incubation at 37 °C in a non-CO₂ incubator, the growth medium was replaced with a base medium (Agilent, Santa Clara, CA, USA) containing 1 mM pyruvate, 4 mM glutamine, and 1 mg/mL D-glucose. Through a 24 min time interval, the cells were sequentially treated with 5 µM oligomycin A, 2 µM FCCP, and 1 µM ronstone/antinomycin A. The oxygen consumption rate (OCR) was determined using a Seahorse XFe24 Analyzer in conjunction with the Seahorse XF Cell Mito Stress Test (Agilent, Santa Clara, CA, USA). The experimental data were analyzed using the Wave2.6 control program (Agilent, Santa Clara, CA, USA).