Base medium

Cells were grown on Seahorse 96-well plates (24,000 cells/well) for 24 h. Cells were washed and incubated in base medium (Agilent Technologies, Les Ulis, France) at 37 °C for 1 h. Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR) were measured in real-time with Glycolysis Stress Test Kit and Mito Stress Test Kit respectively using the Seahorse XFe96 Analyser (Agilent Technologies, Les Ulis, France) following manufacturer’s instructions. Data were normalized by protein content that was measured by the DC™ Protein Assay (BIO-RAD, Marnes La Coquette, France).

The oxygen consumption rate (OCR) of MC3T3‐E1 osteoblastic cells was measured with a Seahorse XFe24 analyzer (Agilent Technologies). The cells were seeded on a cell culture microplate for XFe24 Analyzer at a density of 20 000 cells/well the day before the experiments. On the test day, the medium was replaced with a commercially available Base Medium (Agilent) supplemented with 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine. The OCR was measured following the manufacturer's instructions of the Seahorse XF Cell Mito Stress Test Kit (Agilent). The respiratory rates are reported as oxygen flux per 10 000 cells (pmol O₂/min/cell^*10⁴).

The seahorse Bioscience X96 extracellular flux analyzer (Agilent Technologies) was used to measure the rate change of dissolved O₂ in the medium that immediately surrounded adherent cells cultured in the XF96 V3 cell culture microplate (Seahorse Bioscience). Then, HK-2 cells, which were cultured in RPMI 1640 supplemented with 0.5% FBS, were seeded in the XF96 V3 cell culture microplate at 0.8 × 10⁴ cells per well. Afterwards, these cells were washed and incubated in the base medium (Agilent Technologies) at 37 °C for one hour. The oxygen consumption rates (OCR, pmol.min-1) was measured in real-time using a mitochondria stress test kit, according to manufacturer’s instructions. Sequential compound injections, including oligomycin A (1 μM), FCCP (1 μM) and Rotenone/antimycin A (0.5 μM), were applied on the microplate to test the glycolytic activity. The extracellular acidification rates (ECAR) were measured in real-time using a glycolysis stress test kit, according to manufacturer’s instructions. Sequential compound injections, including glucose (25 mM), oligomycin A (1 μM) and 2-DG (50 mM), were applied on the microplate to test the glycolytic activity.