Fig. 5

ERO1 regulates reactive oxygen species (ROS) production, and inhibition or overexpression of ERO1 can respectively decrease or increase ROS levels. a 143B and SJSA cells were cotreated with EN460 (8 μM) and different concentrations of CYT997 for 24 h, followed by cell proliferation detection using CCK-8 assays. b 143B cells treated with CYT997 (80 nM) and/or EN460 (8 μM) 24 h were analyzed using PI/Annexin V-FITC flow cytometry. Histograms indicate the proportion of apoptotic cells from three separate experiments. c 143B cells were treated with CYT997 (80 nM) and/or EN460 (8 μM) for 24 h and 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. d 143B cells were treated with CYT997 (80 nM) and/or EN460 (8 μM) for 24 h, and apoptosis, autophagy and ER stress-related proteins including c-PARP, caspase-4, LC3B, Beclin-1, CHOP and ERO1 were analyzed by western blotting. e 143B cells were transfected with ERO1-encoding vectors and then treated with or without GSK2606414 for 24 h. Protein levels of ERO1 were detected by western blotting. f 143B cells transfected with ERO1-encoding vectors were treated with different concentrations of CYT997 and 5 nM of NAC for 24 h, followed by cell proliferation detection using CCK-8 assays. g 143B cells were transfected with ERO1-encoding vectors and treated with or without GSK2606414 (2 μM) for 24 h. H2O2 levels were then measured using a hydrogen peroxide assay kit. Histograms indicate the fold change in H2O2 concentration relative to the control group from three separate experiments. *P < 0.05, significantly different compared with the control group. # P < 0.05, significantly different compared with the CYT997 treatment group