• Background: p16^INK4a is a cyclin‑dependent kinase inhibitor encoded by CDKN2A. It blocks CDK4/6 activity, keeps Rb protein hypophosphorylated, and triggers G1‑phase cell‑cycle arrest to suppress oncogenic transformation. Its expression rises markedly with age, serving as a canonical biomarker for cellular senescence. Persistent p16 drives irreversible growth arrest of damaged cells, restrains progenitor cell proliferation, and promotes tissue aging. Clearance of p16‑positive senescent cells alleviates multiple age‑related degenerative phenotypes in pre‑clinical models..
• Targeting strategy: The FKBP-caspase 8 fused with EGFP sequence, driven by human p16 promoter is inserted into mouse Hipp11 (H11) locus in B-P16-ATTAC mice.
• Validation: FKBP and EGFP mRNA was only detectable in homozygous B-P16-ATTAC mice but not in wild-type mice.

AP20187 treatment significantly reduced the elevated p16 expression in aged mice, confirming the successful elimination of senescent cells. In contrast, no obvious changes in p16 expression were observed in 4-month-old young mice following AP20187 intervention.

AP20187-mediated senescent cell clearance is highly specific to aged-associated p16-positive senescent cells, with no detectable effect in young mice lacking substantial senescent cell accumulation.

• Application: B‑P16‑ATTAC mice are a transgenic senescent‑cell ablation model. Upon AP20187 injection, FKBP dimerization activates caspase‑8 and triggers apoptosis of senescent cells, while EGFP enables senescent‑cell tracing. This model is widely used to dissect causal roles of p16‑positive senescent cells in natural aging, age‑related organ degeneration, tumorigenesis and metabolic disorders

Gene targeting strategy for B-P16-ATTAC mice. The FKBP-caspase 8 fused with EGFP sequence, driven by human p16 promoter is inserted into mouse Hipp11 (H11) locus in B-P16-ATTAC mice.

FKBP and EGFP mRNA expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-P16-ATTAC mice by RT-qPCR. Brain, eyes and skeletal muscle RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-P16-ATTAC mice (H/H) (male, 28-week-old, n=3), then cDNA libraries were synthesized by reverse transcription, followed by PCR with FKBP and EGFP primers. FKBP and EGFP mRNA was only detectable in homozygous B-P16-ATTAC mice but not in wild-type mice.

Specific clearance of p16-positive senescent cells by AP20187 treatment in aged B-P16-ATTAC mice. Female homozygous B-P16-ATTAC mice aged 4 months or 18 months were treated via intraperitoneal injection with vehicle or AP20187 (0.2 mg/kg) biweekly for 24 doses across 12 weeks. All animals were harvested at the 12-week endpoint. Eye, inguinal fat pad and dorsal skin was collected for RT-qPCR analysis. p16 expression was markedly upregulated in 18-month-old aged mice relative to young 4-month-old mice. AP20187 treatment significantly reduced the elevated p16 expression in aged mice, confirming the successful elimination of senescent cells. In contrast, no obvious changes in p16 expression were observed in 4-month-old young mice following AP20187 intervention. These findings indicate that AP20187-mediated senescent cell clearance is highly specific to aged-associated p16-positive senescent cells, with no detectable effect in young mice lacking substantial senescent cell accumulation. Values are expressed as mean ± SEM.