Starvation Conditions for Cell Metabolism

Glutamine starvation was described previously [16 (link)]. Briefly, cells were washed once with PBS and cultured in complete medium or glutamine-free medium. For the glucose starvation experiment, DMEM without glucose (11966; Invitrogen, Carlsbad, CA) was supplemented with 10% dialyzed FBS (Gemini) to make glucose-free medium. Glucose (Sigma) was added back to the glucose-free medium to make the complete medium (25 mM) or low-glucose medium (1 mM). For the amino acid starvation experiments, DMEM Ham’s F-12 medium without amino acids (D9811-01; US Biological, Salem, MA) was supplemented with 10% dialyzed FBS. Different amino acids (purchased from Sigma) were added back to amino acid–free medium to make a medium without glutamine, serine, asparagine, aspartate, arginine, leucine, or glycine. To make medium without essential amino acid, the amino acid-free medium was supplemented with nonessential amino acid solution (Sigma M7145) and 4 mM L-glutamine.