Fig 2: Development and validation of functional assay.(A) Western blot of whole cell lysates from PANC-1 cells and PANC-1 cells stably expressing wild type CDKN2A (p16INK4A) or selected pathogenic variants using anti-CDKN2A and anti-vinculin antibodies as a loading control. No expression of CDKN2A (p16INK4A) detected in PANC-1 cells or PANC-1 cell stably expressing empty vector. CDKN2A (p16INK4A) expression detected in PANC-1 cells stably expressing pathogenic variants. (B) Growth of PANC-1 cell and PANC-1 cells stably expressing wild type CDKN2A cDNA (p16INK4A) or selected pathogenic variants over 14 days in culture. Cell proliferation values are shown as mean (n = 3) ± s.d. normalized to PANC-1 cell stably expressed with empty vector. Significant growth inhibition in PANC-1 cell stably expressing wild type CDKN2A (p16INK4A). *p < 0.01. Original files of the full raw unedited blots and the uncropped blots with the relevant CDKN2A and vinculin bands presented in Figure 2—source data 1, Figure 2—source data 2 and 3. For raw data in Figure 2, please refer to Figure 2—source data 4. Figure 2—source data 1.Original file of the full raw unedited CDKN2A blot. Figure 2—source data 2.Original file of the full raw unedited vinculin blot. Figure 2—source data 3.The uncropped blots with the relevant CDKN2A and vinculin bands labelled. Figure 2—source data 4.Raw data in Figure 2B.

Fig 3: Supporting data for Figure 6 AQuantification of mean number of TAF in hepatocytes as a function of the distance from a Ly6G labeled neutrophil. Data are from five mice aged 29 months of age, line indicates linear regression (linear regression and F test: r2 = 0.06; P = 0.0002).BMean number of TAF (left) and mean number of ?H2A.X foci (right) in aged (28–29 months old) INK-ATTAC mice treated with vehicle (VEH) or AP20187 (AP). Data are mean ± SEM of n = 5–6 mice per age group.CComparison between p21 mRNA levels of young (3 months old) and old INK-ATTAC mice (28–29 months old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5–8 mice per age group.D(left) Heat map demonstrating the distribution and relative intensity of the cell surface markers used in the clustering analysis in CyTOF experiment and (right) heat map showing the relative abundance of each cluster for each mouse (5 young and 5 old).EGraph showing relative distribution of different intrahepatic leukocytes in young and old mice.F(left) UMAP plots representing single-cell RNA-sequencing data obtained from (Ma et al, 2020) depicting different population in rat livers (5 and 27 months old). Neutrophils are labeled in red. (right) Violin plots show that p16Ink4a expression is very low in neutrophils at either age group.G–JPure LPS was administered by intraperitoneal injection to wild-type mice (8–10 weeks old; C57BL/6) at a dose of 300 µg/animal for 24 h: (G) Number of neutrophils infiltrates (NIMP+ve) in liver per field of view; (H) mean number of TAF and (I) % of TAF in hepatocytes 24 h following LPS injection; (J) representative images of Immuno-FISH using antibodies against ?H2A.X and Cy-3-labeled telomere-specific (CCCTAA) peptide nucleic acid probe. Data are mean ± SEM of n = 4–5 mice per group. Data information: Statistical analysis was performed using one-way ANOVA (Holm–Sidak method) for multiple comparisons and two-tailed t-test for single comparisons. **P < 0.01; *P < 0.04.

Fig 4: p16Ink4a-expressing cells recruit neutrophils during aging ARNA-sequencing data showing expression of genes associated with neutrophil recruitment during liver aging.B, CQuantification of (B) neutrophil infiltrates (NIMP-positive) and (C) mean number of TAF-positive hepatocytes in liver of young (3 months) and old (24 months) mice. Data are mean ± SEM of 3–5 mice per group.DQuantification of mean number of TAF in hepatocytes as a function of the distance from a Ly6G labeled neutrophil. Data are from five mice aged 29 months of age; line indicates linear relationship between mean TAF and distance (linear regression and F test: R 2 = 0.35; P < 0.0001).ERepresentative micrograph showing hepatocyte from aged mouse containing a TAF located in close proximity to a neutrophil (labeled with Ly6G).FSchematic depicting experimental design.G% of neutrophil migration toward conditioned media (CM) obtained from young (early passage) and replicatively senescent human fibroblasts. Media alone and fMLP were used as negative and positive controls respectively. Data are mean ± SEM of three independent experiments.HSchematic depicting experimental design for panels I–K.IComparison between p16Ink4a mRNA levels of young (3 months old) and old INK-ATTAC mice (28–29 months old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5–8 mice per age group.J% of TAF-positive hepatocytes in old INK-ATTAC mice (28–29 months old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5–6 mice per group.KLiver neutrophils (Ly6G) in old INK-ATTAC mice (28–29 months old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5–7 mice per group.LIntrahepatic leukocyte profiling by mass CyTOF. CyTOF was performed on young (6 months) and old (24 months) INK-ATTAC mice (n = 5 mice per group); 23 unique clusters of intrahepatic leukocytes were defined by a 24-cell surface marker panel using the Rphenograph clustering algorithm and were visualized on a tSNE plot (t-distributed stochastic neighbor embedding plot).M(left) tSNE plots comparing the distribution of young and old intrahepatic leukocytes. Red indicates high frequency categorization of cells to a cluster while blue indicates low frequency; (right) tSNE plots comparing p16Ink4a expression in young and old intrahepatic leukocytes. Red indicates high p16Ink4a expression while blue indicates low expression. Data are from n = 5 mice per group.N% of p16Ink4a-positive cells (detected by CyTOF) in different intrahepatic leukocyte populations isolated from young and old mice. Data are mean ± SEM of n = 5 mice per group.OSchematic depicting experimental design for panels P and Q. 3-month-old wild-type mice were injected with murine Cxcl1 for 7 days.PQuantification of liver neutrophils (Ly6G) in mice injected with vehicle or Cxcl1. Data are mean ± SEM of n = 7–10 mice per group.QQuantification of the % of TAF-positive hepatocytes in mice injected with vehicle or Cxcl1. Data are mean ± SEM of n = 6–7 mice per group.RCorrelation between TAF-positive hepatocytes and neutrophils (linear regression and F test: r 2 = 0.70; P = 0.0001). Data information: Statistical analysis was performed using one-way ANOVA (Holm–Sidak method) for multiple comparisons and two-tailed t-test for single comparisons. **P < 0.01; *P < 0.04.

Fig 5: Neutrophils induce a DNA damage response in bystander cells A, BEffect of neutrophil and recombinant catalase co-culture on (A) mean number of ?H2A.X foci per cell and (B) % tail intensity measured by Alkaline Comet assay. Data are mean ± SEM of n = 3 independent experiments.C–EEffect of hTERT expression (8 and 20 days after primed neutrophil co-culture) on (C) number of 53BP1 foci, (D) expression of p16INK4A and p15 and (E) p16INK4A mRNA levels. Data are mean ± SEM of n = 2–3 independent experiments.FRepresentative micrograph showing nuclear localization of hTERT 4 days after co-culture with primed neutrophils. Data information: Statistical analysis was performed using one-way ANOVA (Holm–Sidak method) for multiple comparisons and two-tailed t-test for single comparisons. **P < 0.01; *P < 0.04.