Fig. 4.

Acute fisetin treatment reduces senescent cell burden in aged wild-type mice and human explants. (A) 22–24-month-old WT C57BL/6 mice were given fisetin (100 mg/kg) or vehicle for 5 days by oral gavage. 72 h after the final dose, the mice were sacrificed and the SA-β-gal⁺ cells were quantified in inguinal fat (n = 6–7 mice per group). Two-tailed unpaired Student's t-test, **p < .01. (B) Schematic diagram of the INK-ATTAC transgene [25]. Expression of FLAG-tagged FKBP-Caspase-8 protein is driven by the p16^Ink4a promoter enabling detection of p16-expressing cells in tissues using immunodetection of FLAG. (C) Aged INK-ATTAC male mice (22–24 months) were acutely treated with fisetin as described above and CyTOF analysis used to quantify p16^Ink4a/FLAG⁺ cell populations in subcutaneous fat tissue (c-kit⁺ mesenchymal stem cells, CD4⁺ and CD8⁺ T cells, NK-1.1⁺ NK cells, and CD146⁺CD31⁺ for endothelial cells). Subcutaneous fat tissue from 6 month-old male mice was used as a control. (D) Quantification of another marker of cellular senescence in the same cell populations (CENP-B⁺ cells). The data are plotted as the mean ± SEM based on n = 9 mice per group. One-way ANOVA with Tukey's multiple comparison test. (E) Human adipose tissue explants (n = 3) were treated with 20 μM fisetin for 48 h, then washed and placed in fresh media for 24 h in order to condition the media. The adipose tissue explants were then stained to measure the percent of SA-ß-gal+ cells. (F) Cytokine and chemokine levels were measured in the conditioned media from the adipose tissue explants using multiplex protein analyses and normalized to adipose tissue weight (n = 3 biological replicates). The results are plotted as the percent expression of various cytokines relative to samples from the same individual treated with vehicle only. Two-tailed paired Student's t-test. Values represented as the mean ± SEM. *p < .05, **p < .01, ***p < .001.