E3024

Cell culture of standard MCF-7 and MDA-MB-231 cells as well as transient transfection assays were performed essentially as described previously (Dardenne et al. 2012 (link); Samaan et al. 2014 (link)). Sequences of siRNAs and TOSS are provided in Supplemental Table S1. AKT activation experiments were performed as follows: 24 h after siRNA transfection, cells were first starved in serum-free medium (Earle's Balanced Salts with Sodium medium, Sigma E3024 and E2888) for 16 h and then reactivated in medium containing 5 µg/mL of SC79 (pan-AKT activator by phosphorylation; S7863, Selleckchem) for 1 h.

Livers of 10-week-old male B6 or C3H mice were perfused with a 37 °C control (EBSS, E3024, Sigma, + 0.1% BSA) or insulin solution (10 nm insulin, Actrapid, in EBSS + 0.1% BSA) at flow rate of 100 ml/h by inserting a 27-gauge syringe in the portal vein and cutting open the right atrium of the heart. After 1 h, the liver was dissected into pieces that were immediately frozen in liquid nitrogen and stored at −80 °C for Western blot analysis.

Amino acid depletion experiment was conducted in 20:80 mixture of normal RPMI-1640 (10% FBS + 2 mM l-glutamine) with Eagle’s balanced salt solution (E3024; sigma) supplemented with 10% FBS. Cells were treated with FK866, JPH203, L-Asp, and indicated combinations for 24 h in the present or absent of amino acid supplementation including 2 mM l-glutamine (Sigma), 0.25 mM l-tryptophan (Sigma), 1× essential amino acid mixture (EAA) (Sigma), and 1× non-essential amino acid (NEAA) (Sigma). Samples were lysed in RIPA lysis buffer supplemented with Protease Inhibitor Cocktail (Thermo Scientific). Equal amounts of proteins were separated by SDS–PAGE. The antibodies used were the following: QPRT (TA501520) from OriGene; 4EBP1 (sc-6936), p-4EBP1(Ser 65/Thr 70; sc-12884), EIF4E (sc-9976), p-EIF4E (Ser209; sc-12885), MTOR (sc-8319), NAMPT(sc-130058), GAPDH (sc-32233), and LDHA (sc-137243) from Santa Cruz; NAPRT (ab-211529), EIF2A (ab26197), p-EIF2A (Ser 51; ab32157), and p-MTOR (Ser 2448; ab1093) from Abcam; and AMPKα (2603), p-AMPKα (Thr 172; 2531), ACTIN (3700), and CHOP (2895) from Cell Signaling. ACTIN and GAPDH were used as protein loading control. Densitometric analysis and normalization relative to control was conducted using ImageJ software. A representative western blot of three-independent biological experiments is shown.