Roswell park memorial institute (rpmi)

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′).

The early-passage of a fetal calve kidney (FCK) primary cell culture, established by Experimental Therapy Department, ICCMGR/ Mustansiriyah University, from a normal FCK, was minced and treated with an enzyme (0.02% trypsin with phosphate-buffered saline (PBS) for 20 min at 37 °C, and then filtered through an 80 um mesh. The filtered fluids were then centrifuged, and the precipitated cells were cultured in a tissue culture flask overnight in Roswell Park Memorial Institute medium (RPMI) (USbiological, Salem, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Capricorn-Scientific GmbH, Ebsdorfergrund, Germany), ampicillin (100 µg /ml), and streptomycin (100 µg/ml) (Capricorn-Scientific GmbH, Ebsdorfergrund, Germany). The human cervix carcinoma (Hela cell line) and human rhabdomyosarcoma (RD) cell line were supplied by the same company and maintained in RPMI medium supplemented with 10% FBS (Capricorn Scientific GmbH), ampicillin (100 µg /ml), and streptomycin (100 µg/ml) (Capricorn Scientific GmbH).