Pyruvate

Dulbecco’s modified Eagle medium (DMEM), phosphate-buffered saline (PBS), fetal calf serum (FCS), Hanks’ Balanced Salt solution (HBSS), penicillin/streptomycin, dihydroethdium (DHE), thiazolyl blue tetrazolium bromide (MTT) and spermidine (#85558) were purchased from Sigma-Aldrich (St. Louis, MO, USA). MitoSOX and glutaMax were from Gibco Invitrogen (Waltham, MA, USA). Tetramethylrhodamine, methyl ester, perchlorate (TMRM) and MitoTrackerRed CMXROS were from Thermo Fisher Scientific (Waltham, MA, USA). The ATPlite1step kit was from PerkinElmer (Waltham, MA, USA) and horse serum (HS) was from Amimed, Bioconcept (Allschwil, Switzerland). Seahorse XFp Cell Mito Stress Test Kit, Seahorse XF Calibrant Solution, Seahorse XF DMEM Assay Medium, pH 7.4, glucose, pyruvate and glutamine were obtained from Agilent Technologies (Santa Clara, CA, USA). The RNA extraction kit was from Qiagen (Hilden, Germany); the GoScript™ Reverse Transcription Mix, Oligo and the GoTaq^® Master Mix for real-time quantitative PCR (RT-qPCR) were from Promega (Dübendorf, Switzerland). The blasticidin was from InvivoGen (San Diego, CA, USA).

Cells were seeded in 6-well plates (200,000 cells/well) and transfected appropriately, as described in figure legends. The day before the assay, cells were detached by trypsinisation, counted, and seeded at 10,000 cells/well in 96-well Seahorse XF cell culture plates (Agilent). The sensor cartridges were hydrated overnight with tissue culture grade H₂O in a non-CO2 incubator at 37 °C as per the manufacturer’s instructions. On the day of the assay, H₂O in the sensor plate was replaced with Seahorse XF Calibrant (Agilent) and cells were washed with HBSS and incubated in Seahorse media (Seahorse XF assay medium (Agilent), 1 mM pyruvate (Agilent), 2 mM glutamine (Agilent), 10 mM D-(+)-galactose). Both sensor and cell plates were incubated in a non-CO₂ incubator at 37 °C for 1 h before the assay. The sensor plate was loaded with oligomycin (15 µM for 1.5 µM final concentration in wells; injector A), CCCP (5 µM for 0.5 µM final; injector B), and antimycin A and rotenone (5 µM/5 µM for 0.5 µM/0.5 µM final; injector C). The sensor plate was calibrated before loading cells and running a mitochondrial stress test using a Seahorse XFe96 (Agilent). Following assays, cells were washed and fixed in 1% (v/v) acetic acid in methanol at −20 °C overnight for SRB assays, which were used for normalising data to protein content.