Animals

Both sexes were studied as specified here and in the Figure Legends. Female (n=30) and male (n=30) young adult (4-month-old) C57BL/6 wild-type (WT) mice were studied. More specifically, the four-month-old females of comparable body weights were randomized to undergoing either sham-operated surgery (SHAM, n=15) or ovariectomy (OVX, n=15) and then were sacrificed 8 weeks later (at age 6 months). Similarly, the 4-month-old males of comparable body weights were randomized to undergoing either SHAM (n=15) or orchidectomy (ORCH, n=15) and then were sacrificed 8 weeks later (at age 6 months). Further, from our earlier work reported previously,⁽¹⁰⁾ we used data on young adult (6-month-old) male (n=12) and female (n=15) mice as well as old (24-month-old) male (n=10) and female (n=9) C57BL/6 WT mice obtained from the NIA. Finally, we generated a cohort of young adult (4-month-old) female INK-ATTAC mice (n=51). In the experiments described herein, we used INK-ATTAC transgenic line 3 – the generation and characterization of which has been described.⁽¹⁸⁾ As detailed previously, INK-ATTAC mice were co-developed by the J.M. van Deursen/D. Baker (Mayo Clinic) and J.L. Kirkland/T. Tchkonia (Mayo Clinic) laboratories. A total of 51 four-month-old female INK-ATTAC mice of comparable body weights were randomized to one of three groups (n=17/group): 1) SHAM+Vehicle (4% EtOH, 10% PEG-400, 86% Tween); 2) OVX+Vehicle (4% EtOH, 10% PEG-400, 86% Tween); or 3) OVX+AP20187 (B/B homodimerizer, Clontech, USA; 10 mg of AP20187 per kg body mass [drug/body mass]) administered intraperitoneally [i.p.]) twice weekly for 8 weeks; all four-month-old young adult INK-ATTAC mice were sacrificed at age 6 months. Note that this dosing regimen has been effective in eliminating senescent cells in old INK-ATTAC mice in multiple previous studies.^(11,29,30) All mice were housed within a pathogen free, accredited facility under a 12-hour light/dark cycle with constant temperature (23°C), and had access to water and food (standard mouse diet, Lab Diet 5053, St. Louis, MO, USA) ad libitum. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC), and all experiments were performed in accordance with Mayo Clinic IACUC guidelines.