Figure 8.

AKT/PKB and/or mTORC1 inhibition are not the leading mechanisms of 3-methyladenine-mediated cytotoxicity. (A) Protein extracts of MEFs subjected to growing concentrations of wortmannin either in the presence (+) or absence (–) of BafA₁ for 8 h were analyzed by western blot. Autophagy was evaluated by LC3-II. Naphthol blue (NB) stained membrane served as a loading control. The image belongs to a representative image out of two experiments. (B) Protein extracts of control untreated MEFs (C) or MEFs treated with 10 mM 3-MA (3-MA), 100 μM Wortmannin (Wn), 5 μM SAR (SAR), 0.5 mM Cpd18 (Cpd18) at 6 and 12 h were analyzed by western blot with antibodies against phospho-AKT (p-AKT) (Ser473 and 308), AKT1, p-ULK (Ser757), ULK, p-4E-BP1(Thr37/46), and 4E-BP1. Quantifications are the ratios between the intensity of phosphorylated proteins normalized to unphosphorylated proteins. Naphthol blue (NB) stained membrane served as a loading control. The images belong to a representative experiment out of three independent repetitions. (C) MEFs were treated for 48 h with the drugs at concentrations stated in the panel. The percentage of propidium iodide (PI)-positive cells (dead cells) was determined by flow cytometry. Bar value is the mean ± SEM (n = 3).