Minimal dmem

Oxygen consumption of intact HeLa cells was recorded with the Seahorse XF^e Extracellular Flux Analyzer (Seahorse Biosciences; North Billerica, MA, USA). ~30.000 cells were seeded into each well of a 96-well Seahorse plate 24 h before the experiment. 60 min before the experiment, cells were washed with XF base medium adjusted to pH 7.4 (Minimal DMEM, 0 mM Glucose, 102353–100 from Seahorse Biosciences), placed in fresh XF base medium pH 7.4 with supplements (1 mM pyruvate, 2 mM L-glutamine, and 5.6 mM D-glucose) and incubated at 37 °C before loading into the XF^e Analyzer. Supplements were from Roth. After recording resting respiration in the analyzer, the following chemicals from Seahorse Biosciences were added sequentially to the cells: oligomycin (1 μm), to measure the nonphosphorylating OCR, FCCP (2 μm), to achieve maximal OCR, and antimycin A (0.5 μm) and rotenone (0.5 μm), for determination of the extramitochondrial OCR. For each experiment, three measurements were performed for the resting OCR, three after oligomycin addition, three after FCCP and three after antimycin A plus rotenone with a 2-min interval of recording followed by 2 min of mixing and 2 min of incubation for each measurement.