HEK293T cells grown in RPMI were washed with PBS and starvation was performed by incubating the cells for 50 min in amino acid-free RPMI without serum. Cell were then stimulated for 10 or 20 min by the addition of RPMI containing a two time concentrated solution of amino acids. After stimulation, the final concentration of amino acids in the media was the same as in RPMI. In case of cycloheximide treatment, amino acid-starved cells were stimulated by addition of cycloheximide diluted in amino acid-free RPMI at a final concentration of 25 μg/ml. HeLa cells grown in RPMI were stimulated for 10 or 20 min by the addition of RPMI containing a two time concentrated solution of amino acids and
insulin (1 uM final concentration, Sigma, I9278).
Concanamycin A (sc202111 Santa Cruz) was used at 5μM and
Torin 1 (4247, Tocris Bioscience,) at 250 nM. Amino acid-free RPMI medium powder (R8999-04A, US biological) was complemented with sodium bicarbonate and sodium phosphate, dissolved in water, adjusted to pH7.4 and filtered. RPMI containing a two time concentrated solution of amino acids was obtained by complementing amino acid-free RPMI medium with
RPMI 1640 amino acids solution (R7131, Sigma), adjusted to pH7.4 and filtered.
L-glutamine (59202C, Sigma) was added shortly before usage.
Rebsamen M., Pochini L., Stasyk T., de Araújo M.E., Galluccio M., Kandasamy R.K., Snijder B., Fauster A., Rudashevskaya E.L., Bruckner M., Scorzoni S., Filipek P.A., Huber K.V., Bigenzahn J., Heinz L.X., Kraft C., Bennett K.L., Indiveri C., Huber L.A, & Superti-Furga G. (2015). SLC38A9 is a component of the lysosomal amino acid-sensing machinery that controls mTORC1. Nature, 519(7544), 477-481.
