Fig. 5
Mitochondria are the main source of B5G9-induced ROS overproduction. a B5G9-induced punctate H2DCFDA fluorescence co-localized with mitochondrial tracker. HepG2 cells were treated with B5G9 (6 μM) or 23-HBA (30 μM) for indicated times. Then cells were stained with H2DCFDA (10 μM) and mitotracker (100 nM) for 30 min. The fluorescence was observed by a fluorescence microscope. Original magnifications: 630 ×, scale bar: 10 μm. b-c B5G9 induced mitochondrial ROS overload. HepG2 cells were treated with B5G9 (6 μM) or 23-HBA (30 μM) for indicated times, and then cells were stained with mitoSOX (5 μM) red for 10 min. The fluorescence was observed by a fluorescence microscope (b). Original magnifications: 630 ×; scale bar: 10 μm. The fluorescence was also detected by a microplate reader (c), ** P ≤ 0.01, *** P ≤ 0.001, B5G9 vs control, ## P ≤ 0.01, 23-HBA vs control. d B5G9-induced elevated ROS was independent of xanthine oxidase, lipoxygenase, cyclooxygenase, cytochrome p450 or NADPH oxidase. HepG2 cells were pretreated with allopurinol (10 μM), NDGA (10 μM), NS-398 (10 μM), SKF-525A (10 μM) or apocynin (20 μM), followed by B5G9 (6 μM) treatment for 3 h, the H2DCFDA fluorescence intensity was detected by a microplate reader. e B5G9 had no influence on activities of CAT, SOD and GPx. After being treated with B5G9 (6 μM) treatment for 3 h, the activities of antioxidases were measured by detection kits