Figure 3. FoxO1 inhibits mTORC1 activity by elevating Sesn3 expression.

A. Overexpressing of Sesn3 downregulates mTORC1 activity. HEK293 cells were transiently transfected with Sesn3L, Sesn3S, or control plasmid. Forty-eight hour after transfection, total proteins were extracted and subjected to immunobloting with the specified antibodies. B. Sesn3 represses mTORC1 activity in a TSC2-dependent manner. Tsc2+/- and Tsc2-/- cells were transiently co-transfected with Sesn3 and myc-S6K1 plasmids. Twenty-four hour after transfection, total protein extracts were subjected to immunoprecipitation using 9E-10 myc-tag antibody followed by immunobloting with anti-p-S6K1, 9B-11 myc-tag, and anti-Sesn3 antibodies. C, D. Knockdown of Sesn3 attenuates the inhibition of mTORC1 activity by FoxO. Tsc2+/- FoxO1(AAA)-ER cells were transfected with Sesn3 or control RNAi. Forty-eight hour after transfection, cells were treated with 4-OHT and harvested at the indicated time points for protein (C) and RNA analyses (D). mTORC1 activity was deduced from three independent experiments and was quantified by the ratio of pS6K1/S6K1. E. The knockdown of Sesn3 has no effect in Tsc2-/- cells. Tsc2-/- FoxO1(AAA)-ER cells were transfected with Sesn3 or control RNAi. Forty-eight hour after transfection, cells were treated with 4-OHT and harvested at the indicated time points for protein and RNA analyses. F. Activated FoxO1 elevates AMPK activity as measured by pACC. MCF7 cells were infected with either FoxO1(AAA) (A3) adenovirus or control virus (E) for twenty-four hours. Cell lysates were subjected to immunobloting with indicated antibodies. G. The knockdown of AMPKα hindered FoxO1-mediated inhibition of mTORC1 activity without an effect on the increased Akt activity by FoxO1. MCF7 cells stably expressing AMPKα or control shRNA were infected with either FoxO1(AAA) (A3) adenovirus or control virus (E) for twenty-four hours. Whole cell lysates were subjected to immunoblotting with the indicated antibodies.