FIGURE 3.

Fisetin protected ARPE-19 cells against H₂O₂-induced DNA damage and apoptosis. Cells were stimulated with or without 20 μM fisetin for 1 h and then treated with 500 μM H₂O₂ for another 24 h. (A) Levels of total p-γH2AX and actin were determined by immunoblotting using cell lysates. (B) Comet assay was performed to analyze DNA damage. Representative immunofluorescence microscopic images are shown. H₂O₂-induced increases in p-γH2AX expression and comet tail formation were apparently alleviated pretreatment of fisetin. (C,D) Cells were stained with DAPI for nuclear morphological observation under a fluorescence microscope for detection of apoptosis. (C) Representative images of DAPI-stained nuclei are shown. (D) Quantification results of nuclei with apoptotic cells are shown in a graph. H₂O₂-exposed cells displayed typical apoptotic properties such as nuclear shrinkage and fragmentation, whereas H₂O₂-mediated morphological changes were markedly restored by fisetin pretreatment. (E,F) After treatment, flow cytometry with annexin V/PI dual staining was performed. Representative histograms (E) and quantitative analysis (F) are shown. Pretreatment of fisetin significantly reduced the increased frequency of apoptotic cells following H₂O₂. (D,F) The graph represents the mean with SD (n = 3, ^*** p ˂ 0.001, compared with the control group; ^### p ˂ 0.001 compared with H₂O₂ treatment group).