Be13 115e

G55 cells, 786-O-LUC and 786-O-HIF1 cells were plated in pyruvate-free high glucose DMEM supplemented with P/S, HEPES and 10% dialyzed FBS (dFBS, 10,000 Mw cut-off: F0392, Sigma-Aldrich). Then cells were treated with DMOG (0.2 mM) and supplemented as necessary with sodium pyruvate (1 mM: BE13-115E, Lonza) for 24 h. Subsequently, the cells were maintained for 2 h in a 5 mM glucose pyruvate-free DMEM medium (SH30022.01, Cytiva HyClone and 11966025, Gibco) with the corresponding treatments. Then, cell media was replaced with labeling solution (pyruvate-free 5 mM glucose DMEM, without FBS, supplemented with 80 μCi/mmol of 5-³H-Glucose (NET531001MC, PerkinElmer) and the corresponding treatments. Cells were incubated in labeling solution at 37°C for 2 h. Then, the supernatant was transferred to glass vials sealed with rubber stoppers. ³H₂O was captured over 48 h at 37°C in hanging wells containing Whatman paper soaked with H₂O. Radioactivity was quantified by liquid scintillation counting.

To measure metabolites, cells were plated in pyruvate-free high glucose DMEM supplemented with P/S, HEPES and 10% dialyzed FBS (dFBS, 10,000 Mw cut-off: F0392, Sigma-Aldrich). Then cells were subjected to 0.5% hypoxia or treated with DMOG (0.1 mM–1 mM) or metformin (2 mM: D150959, Sigma-Aldrich), and supplemented as necessary with sodium pyruvate (1 mM: BE13-115E, Lonza) or sodium α-ketobutyrate (αKB, 1 mM: K0875, Sigma-Aldrich) for 24 h. Subsequently, the cells were maintained for 2 h in a 5mM glucose pyruvate-free DMEM medium (SH30022.01, Cytiva HyClone and 11966025, Gibco) with the corresponding treatments. Finally, this media was changed to the same 5mM glucose pyruvate-free DMEM media - with the corresponding treatments - and cells and supernatants were collected after 60 min, or 15 min when specifically stated, to measure intracellular metabolites and extracellular lactate. For L-Glutamine-5-¹³C tracer analysis, the 786-O-LUC and G55 cells were incubated as above but with pyruvate- and glutamine-free DMEM (11960044, Gibco) supplemented with 4 mM L-Glutamine-5-¹³C (CLM-1166, Cambridge Isotope Laboratories, Inc.), after which the medium was collected. All the samples collected were quenched in liquid nitrogen and stored at −80°C until they were processed.