Figure 3. Inactivation or silencing of NAC1 expression blunts autophagy in ovarian cancer cells treated with cisplatin.

(A) SKOV3/N130 cells cultured in the presence or absence of doxycycline were treated with the indicated concentrations of cisplatin for 24 h. At the end of treatment, cell lysates were prepared, resolved by SDS-PAGE, and subjected to Western blot analysis of LC3 and p62, respectively. Tubulin was used as a loading control. (B) A2780 and OVCAR3 cells were transfected with a non-targeted RNA or siRNA targeting NAC1, followed by treatment with the indicated concentrations of cisplatin for 24 h. At the end of treatment, LC3 and p62 were examined by Western blot. (C) SKOV3/N130 cells cultured in the presence or absence of doxycycline, or A2780 and OVCAR3 cells with or without silencing of NAC1 expression, were transfected with a GFP-LC3 plasmid, followed by treatment with the indicated concentrations of cisplatin for 24h. Quantitation of GFP-LC3 puncta was performed as described in Fig. 1. The bars are the mean ± S.D. of triplicate; results shown are the representative of three identical experiments. **p < 0.01, t-test. (D) SKOV3/N130 cells cultured in the presence or absence of doxycycline, or A2780 and OVCAR3 cells with or without silencing of NAC1 expression, were treated with the indicated concentrations of cisplatin or vehicle for 24h. At the end of treatment, the cells were harvested by trypsinization, fixed and embedded in spur resin. Ninety nm thin sections were cut and examined at 80 Kv with a JEOL 1200EX transmission electron microscope. Arrows indicate autophagic vacuoles.