Fig. 2: Inhibition of AKT phosphorylation is pivotal to ISO-induced autophagy.
From: Isobacachalcone induces autophagy and improves the outcome of immunogenic chemotherapy
A, B Human osteosarcoma U2OS cells stably expressing GFP-AKT or GFP-AKTR25C were treated serum-deprived overnight, then the cells were treated with recombinant IGF1 (rIGF1, 10 nM) or isobacachalcone (ISO; 25 μM) combined with rIGF1. The membrane translocation of GFP-AKT was detected after 10 min (A), and the intensity of membranous AKT was measured (B) Data are means ± SD of quadruplicates (***P < 0.001 vs. untreated control; ##P < 0.01, ###P < 0.001 vs. DMSO/Ctr; Tukey’s multiple comparisons test). C Serum-deprived U2OS cells were treated with ISO (25 μM) with or without recombinant IGF1 (rIGF1, 10 nM) for 6 h, and parallel immunoblots were performed for detecting pAKT, AKT, pmTOR, mTOR, pS6K, S6K, and LC3-II. β-actin (ACTB) was utilized to ensure equal loading (C). D, E U2OS-GFP-LC3 cells transfected with a plasmid coding for AKTT308D/S473D were treated with ISO (25 μM) or torin 1 (300 nM) for 6 h, and GFP-LC3 dots were quantified in (E). Scale bar equals 10 μm. Data are means ± SD of quadruplicates (**P < 0.01, ***P < 0.001 vs. untreated control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. WT; Tukey’s multiple comparisons test). F, G U2OS cells were transfected with a plasmid expressing AKTT308D/S473D. Then the cells were serum-deprived and treated with ISO (25 μM) for 6 h. Parallel immunoblot for pAKT, AKT, and LC3-II were performed, and ACTB was used to ensure equal loading. Band intensities of LC3-II and ACTB were assessed, and their ratio (LC3-II/ ACTB) was calculated (G). Data are means ± SD of three independent experiments (***P < 0.001 vs. untreated control; ###P < 0.001 vs WT; Tukey’s multiple comparisons test).
