Nutrient uptake in Drosophila S2 cells. Representative data obtained from nutrient import experiments performed in triplicate following 72-hour treatments using dsRNA (RNAi) directed against Rheb, TOR, or GFP (control) to deplete the target transcript. (A) Histogram generated (Coulter Counter Z2 software) demonstrating that dsRheb-treated and dsTOR-treated cells are smaller than dsGFP-treated control cells.(B) Bulk amino-acid import normalized to cell number and volume shows that dsRheb-treated and dsTOR-treated cells are not deficient for general amino-acid import. (C) Arginine import normalized to cell number and volume shows that dsRheb-treated and dsTOR-treated cells have increased levels of arginine import. (D) Cellular 2-deoxyglucose import, when normalized to cell number, is significantly reduced in cells treated with dsRheb or dsTOR. When 2DG import is normalized using mean cellular volume (E), or total cellular protein content (F), 2DG import in dsRheb-treated or dsTOR-treated cells is comparable with controls. G) Effects of insulin signaling on 2DG import. Data represent percentage of control 2DG import at 25 (insulin experiments) or 35 minutes after 2DG addition. For the first six bars, growing subconfluent cells were deprived of serum for the indicated periods, and then stimulated with 10 μg/mL bovine insulin for 5 minutes prior to addition of 2DG. Wortmannin (Wort), LY294002 (LY), and 1L-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (AKT-Inh) were added to cells at the indicated doses 30 minutes before addition of 2DG, in the presence of serum. dsRNA directed against Akt (AKTi), PI3K (PI3Ki), or Glut1 (Glut1i) was applied 72 h beforre 2DG addition, also in the presence of serum. Error bars represent standard deviations of duplicate assays.