Fig. 4

Shikonin-induced autophagy in A549 cells. a Cells were treated with 3 or 6 µM shikonin for 3 h, and the levels of the indicated proteins were analyzed by western blotting. Representative images from more than three independent experiments are shown. b Acridine orange staining showed lysosomal (orange) staining in all cells. Acidic lysosomes in shikonin-treated cells, as observed by confocal microscopy, indicate potential lysosomal activation. c Cells were transfected with the GFP-LC3 plasmid for 6 h and then incubated with 3 or 6 µM shikonin for 3 h before analysis by confocal microscopy. Representative images of GFP-LC3-stained shikonin-treated cells are shown (×60). A punctuate pattern of GFP-LC3B indicates autophagosome formation, as observed by confocal microscopy. d Western blot analysis was performed using antibodies against GFP. Immunoblots are representative of at least two independent experiments