Figure 5.

Autophagy enhances DHA-induced apoptosis and inhibition of autophagy partially prevents DHA-induced apoptotic cell death. (A–C) Induction of autophagy by PFTα enhances DHA-induced apoptosis. SiHa cells were incubated for 2 h with or without 20 µM PFTα before incubation for 24 h with 50 µM DHA. The percentage of cells in the Sub G₁ phase (A) and caspase-3 activity (B) were measured as described in the Materials and Methods section. Each bar represents the mean of more than four determinations repeated in three separate experiments. *p < 0.05. The levels of PARP, LC3 and actin were assessed by protein gel blotting (C). (D) Inhibition of autophagy by MG132 attenuates DHA-induced apoptosis. SiHa cells were incubated for 1 h with or without 0.5 µM MG132 before incubation for 24 h with 50 µM DHA. The levels of PARP, p53 and actin were assessed by protein gel blotting. (E–G) Inhibition of autophagy by 3-MA prevents DHA-induced apoptosis. SiHa cells were pretreated with 10 mM 3-MA for 1 h and then incubated with 75 µM DHA for 5 h. Caspase-3 activity (E) and the percentage of Sub G₁ fraction (G) were analyzed as described in the Materials and Methods section. Each bar represents the mean of more than four determinations repeated in three separate experiments. *p < 0.05. The levels of PARP, LC3 and actin were assessed with a protein gel blot assay (F). (H) SiHa cells were seeded in six-well plates. After 24 h, the cells were treated with nontargeting control siRNA (siNC), ATG5 siRNA (siATG5) and ATG7 siRNA (siATG7). At 36 h after transfection, ATG5 and ATG7 mRNA expression levels were examined by RT-PCR analysis. The data shown are representative of three similar ones analyzed by agarose gel electrophoresis after RT-PCR. (I) SiHa cells were seeded in six-well plates. After 24 h, the cells were treated with siNC, siATG5 and siATG7. At 36 h after transfection, cells were treated for 24 h with 50 µM DHA. Light microscope images were captured (left) and cells were harvested and protein gel analysis was performed using the following antibodies: PARP, LC3 and actin as a loading control (right). The data shown are representative of two independent experiments with similar results.