Penicillin streptomycin supplement

HeLa M cells (provided by P. De Camilli, Yale University, New Haven, CT) and HEK293FT cells (Thermo Fisher Scientific) were grown in DMEM (high glucose, with l-glutamine), 10% FBS, and 1% penicillin/streptomycin supplement (all from Invitrogen). Where indicated, cells were starved by incubation in amino acid–free RPMI (US Biological) for 2 h. Amino acid refeeding was achieved by adding 1× MEM amino acid supplement (Invitrogen) into RPMI. Plasmid transfections were performed with 0.1 µg plasmid DNA, 0.3 µl Fugene 6 transfection reagent (Promega), and 20 µl Opti-MEM (Invitrogen) that was added to 500 µl media containing 20,000 cells per well in 24-well plates. siRNA transfections were performed with 7.5 µl 20 µM siRNA, 5 µl RNAiMAX transfection reagent (Invitrogen), and 500 µl Opti-MEM (Invitrogen) that was added to 2 ml media containing 120,000 cells per well in six-well plates. Experiments were performed 2 d after transfections. For transfection of larger dishes, the volumes of reagents and the number of cells were increased proportionally to surface area of the dish. Control siRNA was purchased from Integrated DNA Technologies (5′-CGUUAAUCGCGUAUAAUACGCGUAT-3′), and RagC siRNA was purchased from GE Healthcare (5′-GCAAUUAUCAAGCUGAAUA-3′).

HEK293FT (Life Technologies, Carlsbad, CA) and HeLa M cells (provided by P. De Camilli, Yale University, New Haven, CT) were grown in high-glucose DMEM (+l-glutamine), 10% fetal bovine serum, and 1% penicillin/streptomycin supplement (Invitrogen, Carlsbad, CA). Where indicated, cells were starved for 90 min by incubation with amino acid–free RPMI media (US Biological, Swampscott, MA). Amino acid refeeding was performed with 1× MEM amino acid supplement (Invitrogen) added to starvation medium for 15 min. Transfections were performed with 500 ng of DNA, 100 μl of OptiMEM (Invitrogen), and 1.5 μl of FuGENE 6 transfection reagent (Promega, Madison, WI) per 35-mm dish and scaled up proportionately for larger and smaller dishes. siRNA transfections were performed with 5 μl of RNAiMAX (Invitrogen), 500 μl of OptiMEM (Invitrogen), and 7.5 μl of 20 μM siRNA stock suspended in 100 mM KAc and 30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5. We used 250,000 HEK 293FT cells per 35-mm dish for siRNA transfection and incubated them for 2 d posttransfection before experiments. siRNA sequences purchased from Integrated DNA Technologies (IDT, Coralville, IA) are summarized in Supplemental Table S1.