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. 2009 Mar 20;284(12):8023–8032. doi: 10.1074/jbc.M900301200

FIGURE 3.

FIGURE 3.

Torin1 inhibits mTORC1-dependent processes that are resistant to rapamycin. A, Torin1 but not rapamycin (Rapa) causes LC3 to relocalize from the cytoplasm to autophagosomes. Wild-type (p53-/-) or Rictor-null (p53-/-) MEFs were transiently transfected with GFP-LC3 and treated with vehicle (Veh) (DMSO), 50 nm rapamycin, or 250 nm Torin1 for 3 h before being fixed and processed. Cells were also stained with Hoechst to visualize nuclei and imaged at ×63. B, amino acid starvation and Torin1, but not rapamycin, cause LC3 degradation. Wild-type (p53-/-) and Rictor-null (p53-/-) MEFs were treated with vehicle (DMSO), 50 nm rapamycin, 250 nm Torin1 or grown in amino acid (AA)-free conditions for 0, 1, 3, or 6 h. Cells were lysed at the indicated time points and analyzed by immunoblotting. Induction of autophagy causes the degradation of the native LC3B (LC3B-I) protein and the transient accumulation of the faster running lipidated version (LC3B-II). C, Torin1 suppresses global protein synthesis through a rapamycin-resistant and mTORC2-independent process. Wild-type (p53-/-) and Rictor-null (p53-/-) MEFs were treated with vehicle (DMSO), 50 nm rapamycin (Rap), 250 nm Torin1, or 10 μg/ml cycloheximide (Chx) for 2.5 h and then pulsed with 35S-labeled methionine and cysteine for 30 min. The amount of 35S incorporation was determined by scintillation counting. Measurements were made in triplicate, and error bars indicate standard deviation.