Starvation protocol

For amino acid stimulation studies, sub-confluent cells were serum-starved for 16 h followed by amino acid starvation for 4 h prior to stimulation with a 10× mixture of total amino acids for 30 min, unless otherwise indicated. The final concentration of amino acids used to stimulate the cells was 2×. The 10× mixture of total amino acids was prepared from individual powders of amino acids^9,12. For amino acid deprivation studies, complete growth medium was replaced by dialyzed serum for 4 h. The recipe of the 10× mixture of total amino acids is provided in Supplementary Table ¹. Chemical treatments were performed with indicated drugs for the last 2 h of amino acid starvation.

All the dry/powder amino acids were added to 500 ml of dH₂O. Another 476 ml of dH₂O was added to make final volume 1 l. Solution was mixed thoroughly by shaking and heating (mild) on a stir plate followed by filter sterilization using 0.2 micron filtration units.