Figure 3.

Amino acid-induced activation and lysosomal localization of mTORC1 is prevented by endocytosis inhibition. A, 293T cells were starved for amino acids in amino acid-free RPMI1640 medium containing 10% dialyzed Nu-Serum in combination with 0.1% DMSO or 80 μm Dynasore. After 50 min starvation, cells were stimulated for 40 min with amino acids by adding 1/50 volume of ×50 amino acid solution containing l-glutamine. B, quantification of immunoblot data in A. Mean values of two independent experiments are shown. C, 293T cells were treated as in A and subjected to immunofluorescence with anti-mTOR and anti-Lamp1 antibodies. Scale bar = 10 μm. D, quantification of cells that exhibit lysosomal localization of mTOR by analysis of image data in C. Each bar indicates mean ± S.D. of three independent experiments. ***, p < 0.001. E, quantification of fluorescence intensity of mTOR on lysosomes by analysis of image data in C. Each bar indicates mean ± S.D. of three independent experiments. ****, p < 0.0001.