Tryptophan Starvation of HeLa Cells

Trp sarvation of HeLa cells were induced as previously reported [38 (link)]. In detail, HeLa cells were seeded in 60 mm dishes (Corning) and incubated for 24 h. Cells were washed (3×) in PBS and incubated for 24 h in amino acid-free DMEM medium (high glucose) (#048-33575; FUJIFILM Wako Pure Chemical Corp.) supplemented with 10% (v/v) US dialyzed FBS (GE Healthcare Life Sciences), 2 mM Gln, and amino acids (0.4 mM arginine, 0.2 mM cysteine, 0.4 mM glycine, 0.2 mM histidine, 0.8 mM isoleucine, 0.8 mM leucine, 0.8 mM lysine, 0.2 mM methionine, 0.4 mM phenylalanine, 0.4 mM serine, 0.8 mM threonine, 0.4 mM tyrosine, 0.8 mM valine). Note, with the exception of Trp, the concentration of each of the amino acids corresponds to that of standard DMEM (#10313-021; ThermoFisher Scientific).