Figure 4: BjAnn1 exhibits ROS scavenging activity and modulates the expression of genes involved in oxidative stress and antioxidant defense. (A) U251 and U87 cells were transfected with EV- and BjAnn1 plasmids and after 48 h cells were seeded in 96-well plates (1×104/well) and ROS levels were determined using total ROS/ Superoxide Detection Kit following manufacturers’ instructions. (B) Potential changes in the multiple gene expression profile in BjAnn1-expressing U87 cells were determined in comparison to EV-treated cells using Human Oxidative Stress and RT2 Profiler Human Antioxidant Defense arrays as explained in Materials and Methods. The relative fold changes values of gene expression in BjAnn1-expressing cells in comparison to EV-treated controls were obtained from three experimental replicates and represented in the form of a heat map. (C) Whole cell lysates were prepared from control (EV) and BjAnn1 expressing U251 and U87 cells and the expression levels of SOD2 and SOD3 were determined by western blotting. (D) U251 and U87 cells transfected with EV- and BjAnn1 plasmids were seeded in 96-well plates (1×104/ well) and treated with 10 μM H2O2 for 2 hours, then fresh medium was added and further incubated for another 12-16 hours. Cells were incubated in 100 μl/well of ROS/ Superoxide detection mix for 1 h at 37°C. Total ROS content was determined by reading culture plates with a fluorescence microplate reader at 488 nm-530 nm.