Oxidative Medicine and Cellular Longevity / 2016 / Article / Fig 5

Research Article

Hericium erinaceus Inhibits TNF-α-Induced Angiogenesis and ROS Generation through Suppression of MMP-9/NF-κB Signaling and Activation of Nrf2-Mediated Antioxidant Genes in Human EA.hy926 Endothelial Cells

Figure 5

HE downregulates NF-κB nuclear translocation through the suppression of I-κB degradation in TNF-α-activated EA.hy926 cells. (a) Western blot was performed to monitor the cytosolic I-κBα and nuclear p65 protein expressions. Cells were treated with indicated concentrations of HE (50–100 μg/mL) for 2 h in the presence or absence of TNF-α (10 ng/mL) for 1 h. An equal amount (50 μg) of cytosolic or nuclear lysate from each sample was resolved by 12% SDS-PAGE with β-actin as a control. Relative changes in protein bands were measured using densitometric analysis. (b) Immunofluorescence staining shows the changes of nuclear translocation of NF-κB. Cells were grown in chamber slides that were exposed to HE (100 μg/mL) for 2 h in the presence or absence of TNF-α (10 ng/mL) for 1 h, fixed, and permeabilized. Then the cells were incubated with anti-P65 antibody followed by FITC-labeled secondary antibody. Cells were stained with DAPI (1 μg/mL) for 5 min and examined by fluorescence microscopy (magnification ×200). Results are presented as mean ± SD of three independent assays; indicates significant difference compared to control, and indicates significant difference compared to TNF-α alone treatment.