Analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was performed using a Seahorse extracellular flux XF96 analyser (Seahorse Bioscience, Billerica, MA) in accordance with manufacturer's instructions. Briefly, cells were seeded at a density of 1.5 × 104 cells per well in Matrigel-treated extracellular flux 96-well culture microplates in 150 μl of corresponding cell culture media and incubated overnight at 37 °C. The next day, before analysis, culture media was replaced with 150 μl of unbuffered assay media and cells were incubated for 1 h at 37 °C for pH and temperature stabilization. Analysis of OCR and ECAR was performed simultaneously both at basal conditions and after injections of the inhibitors in the XF Cell Mito Stress Test Kit (1 μg ml−1 Oligomycin, 0.5 μM FCCP, 1 μM Antimycin A+1 μM Rotenone (Sigma)). To evaluate the effect of the respective PI3K and MAPK inhibitors LY294002 and PD0325901 on cellular metabolism, cells were incubated overnight (12 h) in the presence of 10 μM LY294002 or 1 μM PD0325901. The next day, before analysis, culture media was replaced and analysis
