Fig. 2.

Akti-1/2 upregulates autophagy in ascites-derived ovarian cancer cells. (A) iOvCa147-E2 or EOC samples were incubated with increasing [Akti-1/2] in complete medium (10% fetal bovine serum) for 24h. Lysates were obtained and immunoblot was performed for indicated proteins (representative blots of three experiments with iOvCa147-E2 and of five independent EOC samples). (B) iOvCa147-E2 or EOC samples were seeded to glass coverslips and subsequently incubated with Akti-1/2 (20 μM) or DMSO control in complete medium (10% fetal bovine serum) for 24h. Indirect immunofluorescence was performed using anti-LC3 antibody and nuclei were stained with 4′,6-diamidino-2-phenylindole. Scale bar, 50 μm. (C) Transmission electron microscopy was performed on EOC67 cells treated with Akti-1/2 or DMSO as indicated. Insets of Akti-1/2-treated cell images (i–iii) indicate autolysosome structures in the cytoplasm. Scale bar, 500nm; n, nucleus. (D) Cells in 6-well plates were treated with Akti-1/2 (EC50) ± CQ (50 μM) and lysates obtained 24h post-treatment, and immunoblot was performed for indicated proteins. Representative blots of iOvCa147-E2 and EOC129 are depicted. Quantification of LC3II expression relative to actin are shown, as determined using the Chemidoc System and ImageLab 4.1 software (Biorad). (E) Quantification of LC3-II was normalized to actin for five independent EOC samples and one-way analysis of variance with Tukey’s multiple comparison test was used to compare means. Letters denote statistically significant differences (P ≤ 0.01). Data are represented as mean ± SEM.