Glucose free dmem

All nutrient deprivation experiments were performed in the absence of serum for 24 hours, unless otherwise indicated. Cells were plated in complete culture media, which was exchanged with nutrient-deprived media (described below) 1–3 days after cell seeding. Nutrient-deprived media was not changed throughout the course of the experiments, unless otherwise indicated. For non-essential amino acid deprivation experiments, MEM medium without glutamine (Sigma) was used, and 1X MEM non-essential amino acids (Sigma) and/or 4 mM glutamine (Corning) were added for the corresponding controls. For cystine deprivation experiments, DMEM medium without methionine, cystine and glutamine (Sigma) was used, and 0.030 g/L methionine (Sigma), 0.063 g/L cystine (Sigma) and/or 4 mM glutamine were added for the corresponding controls. For glutamine deprivation experiments, glutamine-free RPMI (Corning), glutamine-free DMEM (Corning) or glutamine-free IMDM (Sigma) was used. For leucine deprivation experiments, leucine-free RPMI (Crystalgen) or leucine-free DMEM (Crystalgen) was used. For glucose deprivation experiments, glucose-free RPMI (Crystalgen) or glucose-free DMEM (Corning) was used. In Figures S3D, S4A and S4B, SW1990 cells were starved of glutamine in the presence of dialyzed FBS for 24 hours (S4A and S4B) or 48 hours (S3D).

COS1 cells were plated in triplicate at a density of 3 × 10⁴ cells/ well in black clear bottom 96-well microplates (Corning) and allowed to become adherent in complete media overnight at 37 °C in a humidified atmosphere of 5% CO₂. The following day, the cells were gently washed with PBS (3×) and serum starved in glucose free DMEM (Corning) for 4 h at 37 C, 5% CO₂. The cells were then treated with the RBPs for 1 h at 37 °C. Media containing RBPs was aspirated and glucose uptake was assessed with a Glucose Uptake Cell-Based Assay Kit (Cayman Chemicals) in which we treated cells with 50 μM 2-NBDG in the presence of 250 nM insulin (Calbiochem) for 15 min at 37 °C in a humidified atmosphere of 5% CO₂. Following stimulation, the cells were gently washed with PBS (2×) before the addition of 100 μL of the cell-based assay buffer. 2-NBDG uptake was measured using an EnVision plate reader (Perkin Elmer) with an excitation wavelength of 485 nm and emission wavelength of 535 nm.