a, Immunofluorescence (IF) detection of p16 and ORF1 in early passage, 3× and senescent cells. b, Representative images of combinatorial ORF1 and p16 or ORF1 and phosphorylated STAT1 (pSTAT1) staining in human dermis. The experiments in a and b were repeated three times independently with similar results. c, Cells were plated on coverslips, stained and quantified as described in the Methods. A total of 200 cells in several fields were scored for each condition. Insets show the percentage of cells found in each quadrant. d, e, Abundance of ORF1 and p16 or pSTAT1 cells in human skin. Skin biopsies were cryosectioned and stained as described in the Methods. A total of 200 dermal fibroblast cells in several fields were scored for each subject. Aggregated data for 4 subjects (800 cells) are shown. f, Data in c and d were recalculated to show the relative abundance of p16+ cells among all cells, and ORF1+ cells in the p16+ pool of cells. g, Data in e were recalculated as in f. h, Characteristics of the human subjects used in the analysis of dermal fibroblasts. These specimens were collected as part of the ongoing Leiden Longevity Study62. The specimens used here were chosen randomly from leftover material. The telomere dysfunction-induced foci (TIF) assay34 relies on a two-parameter (colour) visualization of telomeres (using a FISH probe) and immunofluorescent detection of DNA damage foci (using antibody to 53BP1). Because of limiting material, it was not possible to combine detection of p16 with TIFs in a three-colour experiment.