Figure 3

Fisetin inhibits the nuclear translocation of NF-κB. BV2 microglial cells were treated with the indicated concentrations of fisetin (0–5 µM) for 2 h and subsequently stimulated with 1 µg/mL LPS for 2 h, followed by stimulation with 1 mM ATP (LPS/ATP). (A) Nuclear proteins were extracted after 1 h of treatment with ATP and western blotting was performed. Nucleolin was used as a loading control. Relative densities of p50 and p65 were calculated using ImageJ software. (B) In a parallel experiment, the cells were fixed with 4% paraformaldehyde and immunostained for p65 with Alexa Fluor 647-conjugated secondary antibody. The images of the cells were captured using a CELENA S Digital Imaging System. The results indicate the mean ± standard error median (SEM), and is representative of the results obtained from three independent experiments. ^### p < 0.001 vs. untreated cells; *** p < 0.001 and * p < 0.05 vs. LPS/ATP-treated cells.