Pyruvate assay kit

Extracellular glucose concentrations were determined using the glucose assay kit 1 (#1200032002, Eton Bioscience, USA), and absorbance measured at 490 nm; Extracellular pyruvate concentrations were determined using the pyruvate assay kit (#1200041002, Eton Bioscience), and absorbance was measured at 570 nm; Extracellular glutamine was determined using the Glutamine assay kit (#ab197011, Abcam, Cambridge, UK), and absorbance was measured at 450 nm; Extracellular l-lactate concentrations were determined using the l-Lactate Assay Kit (#MAK329, Merck), and absorbance was measured at 565 nm.
All assays were performed according to manufacturer's instructions and absorbance readings were measured using a POLARstar Omega microplate reader (BMG Labtech). Metabolite concentrations were extrapolated from a linear regression fit of known standards, and then normalised to CyQUANT data (ng/ml DNA) to account for any differences in the number of cells.

IP pathway genes were knocked down for 24 and 48 h, and cells (100 µl) were incubated with a 100 µl CellTiter-Glo luminescent cell viability assay (Promega) to measure ATP using a GloMax 96-microplate lLuminometer (Promega). In parallel, cell supernatants were quantified using a pyruvate assay kit, both according to manufacturer's instructions (Eton Bioscience). Antimycin (8 mM), K cyanide (50 µM), sodium azide (0.5 mM), rotenone (5 µM), oligomycin (50 µg/ml), atractyloside (200 µM), and SHAM (100 µM) were incubated with CN IPMK 24 h after knockdown and ATP was measured at 48 h as described above.