Effect of á-TOS on autophagy in PC3 cells treated with VK3+AA.

(A) PC3 cells were treated with the drugs as indicated and the level of the LC3-II protein normalized to â-actin was expressed as mean ± S.D. of the LC3-II/LC3-I densitometry ratio. (B) PC3 cells were treated with the drugs as indicated for 2 h and assessed for morphology by TEM. Magnification 4,000×, scale bar  = 0.5 µm (upper images), magnification 9,000×, bar  = 0.5 µm; N =  nucleus, m =  mitochondria, a =  autophagosomes. (C) PC3 cells were treated as indicated, and formation of the AO-accumulating acidic vesicular organelles (orange-red fluorescence) was detected using flow cytometry. (D) PC3 cells were treated as indicated in presence or absence of autophagy inhibitors (3MA and CQ), and cell viability was assed after 24 h of incubation. (E) The formation of autophagosomes in control cells (1) and in the presence 3MA (2) and CQ (3) was evaluated by western blotting as the expression of LC3-II after 2 h of drug treatments. The data are expressed as a change with respect to untreated control, and are mean values ± S.D. (n = 3), the images are representative of three independent experiments. * p<0.05, compared with controls.