Collagen coated 24 well cell culture plates

VSMC were seeded in appropriate collagen-coated 24-well cell culture plates (from Seahorse Biosciences, Copenhagen, Denmark) at a cell density of 2 × 10⁴ cells/well and treated as indicated. Cells were then kept in serum and glucose free assay medium (DMEM; pH 7.35–7.40) at 37 °C and ambient CO₂ for one hour before they were subjected to assessment of the extracellular acidification (ECAR in mpH/min) [and oxygen consumption rate (OCR in pmol O₂/min) if needed]. Appropriate test kits came from Seahorse Biosciences, were performed according to the manufacturer's instructions and analyzed on a Seahorse 24XFe extracellular flux analyzer and Wave software (www.seahorsebio.com) as described previously in [16 (link),17 (link)]. Optimized inhibitor concentrations for VSMC were 3 μM oligomycin and 100 mM 2-deoxyglucose (2-DOG). Normalization to cell mass was routinely performed by crystal violet staining after the analysis in order to account for potential differences in cell number. Basal glycolytic activities were taken as readout and were calculated as follows: ECAR upon injection of glucose corrected for non-glycolytic acidification (ECAR in the presence of DOG).

MEF were seeded in appropriate collagen-coated 24-well cell culture plates (from Seahorse Biosciences, Copenhagen, Denmark) at a cell density of 2.7 × 10⁴ cells/well. Cells were kept in serum-free medium (glucose free DMEM plus 2 mM glutamine, 2 mM pyruvate, pH 7.35–7.40) at 37 °C and ambient CO₂ for 1 h before they were subjected to mitochondrial (readout: oxygen consumption rate (OCR) in pmol O₂/min) stress tests. Appropriate test kits came from Seahorse Biosciences, were performed according to the manufacturer’ instructions, and analyzed on a Seahorse 24XF^e extracellular flux analyzer and Wave software (www.seahorsebio.com) as described elsewhere [31 (link)] and in detail in the supplemental information.