Low doses of Rapamycin, Torin 1, and Torin 2 inhibit mTOR and induce autophagy. (A) RAW264.7 cells were pretreated with 1 uM or 10 uM of the mTOR inhibitors indicated and then challenged with 1 ug/ml E. coli derived LPS for 3 hours. Protein lysates were prepared and western blots for total ribosomal S6 and phosphorylated ribosomal S6 are shown. Shown are data representative of two independent assays (B) RAW264.7 cells were infected with M. smegmatis (MOI 5) and treated with the mTOR inhibitors shown. Protein lysates were prepared and western blots for Actin and phosphorylated ribosomal S6 were performed. Shown are data representative of two independent assays (C) A549 cells were treated with 10 uM of the indicated inhibitor for 3 hours and then stained for endogenous LC3B, or an isotype control IgG, and imaged by fluorescence microscopy. Shown are data representative of two independent assays. (D) RAW264.7 cells were loaded with DQ-BSA, either left untreated (−DMSO) or treated overnight with the indicated concentrations of the mTOR inhibitors shown, and analyzed by flow cytometry. Shown is the combined percentage of DQ-BSA positive cells (+/− SEM) and the mean fluorescent intensity (and intensity range) derived from two independent assays with 3 replicates per assay. For analysis of the percent DQ-BSA positive cells, asterisks indicated p < 0.05 for drug treated samples versus untreated.
Zullo et al. BMC Biochemistry 2014 15:4 doi:10.1186/1471-2091-15-4