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

(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 Materials and Methods. 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 (pMaterials and Methods.