GPD induces autophagic cell death in a dose-dependent manner.

<p>(A) GPD induces autophagy in SK-MEL-28 cells whereas Rb1 cannot. GFP-LC3-transfected SK-MEL-28 cells were incubated with or without 40 µM of GPD and compared with Rb1 treatment for 48 h. Cells were fixed with 4% formaldehyde and then incubated with anti-p62/SQSTM1 antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104305#s2" target="_blank">Materials and Methods</a>. Immunofluorescent staining was observed by confocal microscopy. (B) LC3 aggregations in transfected cells increase in the presence of GPD treatment in a dose-dependent manner. GFP-LC3 aggregations were counted by microscopic (×400) observation of the immunofluorescently stained cells. Results are shown as means ±SD. The asterisks (*) and (**) indicate statistical significance (p<0.05 and p<0.001, respectively) compared with untreated control groups. (C) The expression of LC3-II, p62/SQSTM1 and Beclin-1, which are critical autophagic markers, increased in the presence of GPD treatment, but not Rb1 treatment. Cells were treated with or without various concentration of GPD or 40 µM of Rb1 for 48 h, and protein expression was determined by western blot analysis. (D) GPD increased the translocation of cytochrome c from the mitochondria to the cytosol. Mitochondrial fractionation was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104305#s2" target="_blank">Materials and Methods</a>.