Figure 5

Fisetin inhibits mitochondrial membrane depolarization and promotes mitophagy. 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) for an additional 2 h. (A) Populations of depolarized mitochondria were measured using a Muse MitoPotential Kit. (B,C) Cells were stained with 2 µM MitoSOX Red for 10 min and (B) fluorescence intensities were measured using fluorometry. (C) Images of cells were captured using a CELENA S Digital Imaging System. (D) In a parallel experiment, total proteins were extracted after 9 h of treatment with LPS/ATP. Western blotting was performed for detecting the expression of LC3. β-Actin was used as a loading control. (E) Cells were fixed with 4% paraformaldehyde and immunostained for p62 with Alexa Fluor 647-conjugated secondary antibody. The results indicate the mean ± standard error median (SEM), and is representative of the results obtained from three independent experiments. Images were captured using a CELENA S Digital Imaging System. ^### p < 0.001 vs. untreated cells; *** p < 0.001, ** p < 0.01 and * p < 0.05 vs. LPS/ATP-treated cells.