Fig. 4

CYT997 induced intracellular reactive oxygen species (ROS) generation, and ROS scavenging decreased CYT997-induced apoptosis, autophagy and endoplasmic reticulum (ER) stress (a-b). Osteosarcoma cells 143B and SJSA were treated with or without CYT997 (80 nM) for 24 h. Cells were stained with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) or 1 μM MitoSOX Red dye at 37 °C in the dark for 30 min and then observed by fluorescence microscopy and confocal microscopy. Scale bars = 50 and 5 μm. c Fluorescence intensity of DCFH-DA was detected using flow cytometry. Histograms indicate the fold change in DCFH-DA intensity relative to the control group from three separate experiments. d 143B and SJSA cells were cotreated with NAC (5 mM) and different concentrations of CYT997 for 24 h, followed by the CCK-8 assay for cell proliferation. e 143B cells treated with CYT997 (80 nM) and/or NAC (5 mM) analyzed using PI/Annexin V-FITC flow cytometry. Histograms indicate the proportion of apoptotic cells from three separate experiments. f 143B cells were treated with CYT997 (80 nM) and/or N-acetyl-L-cysteine (NAC) (5 mM) for 24 h, and apoptosis, autophagy and ER stress-related proteins including c-PARP, caspase-4, LC3B, Beclin-1, p-PERK, p-eif2α, CHOP and ERO1 were analyzed by western blotting. *P < 0.05, significantly different compared with the control group. # P < 0.05, significantly different compared with the CYT997 treatment group