Fig. 2

CYT997 induced autophagy in OS cells, and inhibition of autophagy increased CYT997-induced apoptosis. a Osteosarcoma cell lines 143B and SJSA were transiently transfected with GFP-LC3-encoding plasmids for 24 h, treated with or without CYT997 (80 nM) for 24 h and stained with LysoTracker Red DND-99 (50 nM). Green color represents the formation of autophagosomes, and red color shows cellular acidic compartments, indicative of lysosomes and autolysosomes. Colocalization of autophagosomes and lysosomes was examined by confocal microscopy. Scale bars = 20 μm. b CYT997 induced accumulation of autophagosomes in osteosarcoma cells, as shown in the electron micrographs. Arrows indicate autophagosomes, and arrowheads indicate ER. c Osteosarcoma cells were treated with CYT997 (80 nM) for 24 h. Autophagy-related proteins, LC3B and beclin-1, were analyzed by western blotting. d 143B and SJSA cells were preincubated with 3-methyladenine (3-MA) (5 mM) for 2 h and then treated with CYT997 (80 nM) for 24 h, followed by cell proliferation detection using CCK-8 assays. e Osteosarcoma cells were preincubated with 3-MA (5 mM) and then treated with CYT997(80 nM) for 24 h and analyzed using PI/Annexin V-FITC flow cytometry. Histograms indicate the proportion of apoptotic cells from three separate experiments. f Cells were treated with 80 nM CYT997 and 3-MA for 24 h, and the levels of c-PARP, LC3B and Beclin-1 were assessed by western blotting. *P < 0.05, significantly different compared with the control group. # P < 0.05, significantly different compared with the CYT997 treatment group