The observation that bafilomycin inhibits EGFP-LC3 processing and degradation but that it does not have an effect on the inhibition of mTORC1 signaling by the four active chemicals exhibits that mTORC1 signaling inhibition is not a consequence of stimulation of autophagy and is consistent with stimulation of autophagy lying downstream of mTORC1 inhibition. mTOR is present in two distinctive complexes mTOR intricate 1 which phosphorylates S6Ks, 4E-BPs and PRAS40 and mTORC2 which catalyzes the phosphorylation of PKB/Akt and SGK1. Insulin receptor substrate-one, and to a lesser extent IRS-two, protein stages are regulated by S6K1. Hyperactivation of S6K1 signaling leads to transcriptional inhibition of the IRS-1 gene and degradation of IRS-1 and IRS-two proteins. This is obvious in equally TSC1 and TSC2 null mouse embryo fibroblasts which exhibit reduced insulin receptor/PI3K signaling and PKB/Akt phosphorylation at 1346528-50-4 Ser473 as a result of mTORC1/S6K1 signaling hyperactivation. Extended treatment of cells that display elevated mTORC1/S6K signaling with rapamycin restores PI3K signaling and PKB/Akt phosphorylation on Ser473. We reasoned that other inhibitors of mTORC1/S6K signaling, this kind of as these identified in this display, may possibly also STA-9090 enhance PKB/Akt phosphorylation. As predicted, MCF-7 cells, which exhibit elevated mTORC1 signaling like TSC1 or TSC2 null MEFs, confirmed elevated phosphorylation of Ser473 in PKB/Akt when taken care of with niclosamide, perhexiline, amiodarone or rottlerin. The increase in PKB/Akt Ser473 phosphorylation intently paralleled the decrease in mTORC1 action as a function of concentration for the four chemical compounds. The observation that the four chemical compounds elevated PKB/Akt phosphorylation at Ser473 as an alternative of reducing it displays that they inhibited mTORC1 but not mTORC2 signaling. MCF-seven cells expressing EGFP-LC3 were incubated with perhexiline, niclosamide, rottlerin, or amiodarone for 4 h in full medium, the chemical compounds have been washed away and S6K phosphorylation was measured right away right after washing.