Fig. 3

CYT997 induced endoplasmic reticulum (ER) stress in osteosarcoma cell lines, and inhibition of the ER stress pathway decreased CYT997-induced apoptosis, autophagy and reactive oxygen species (ROS) production. a Osteosarcoma cell lines 143B and SJSA incubated with CYT997 (80 μM) for 24 h were visualized by electron microscopy; arrowheads point to the ER, and arrows indicate autophagosomes. b ER stress-related proteins including PERK, p-PERK, eif2α, p-eif2α, CHOP, and ERO1 were detected by western blotting after incubation of the cells with various concentrations of CYT997 for 24 h or treatment with CYT997 (80 nM) for different durations. c 143B and SJSA cells were cotreated with GSK2606414 (2 μM) and different concentrations of CYT997 for 24 h, followed by cell proliferation detection using CCK-8 assays. d 143B cells treated with CYT997 (80 nM) and/or GSK2606414 (2 μM) were analyzed using PI/Annexin V-FITC flow cytometry. Histograms indicate the proportion of apoptotic cells from three separate experiments. e 143B cells treated with CYT997 (80 nM) and/or GSK2606414 (2 μM) for 24 h, stained with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37 °C in the dark for 30 min. Histograms indicate the fold change in DCFH-DA intensity relative to the control group from three separate experiments. f 143B cells were treated with CYT997 (80 nM) and/or GSK2606414 (2 μM) 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