Figure 9. Increases in senescent stromal cells in aged human skin correlate with increases in inflammation.

(a) IF staining for p16^INK4a (red) in human skin samples grouped by age. Nuclei are DAPI-stained (blue). Scale bar, 100 or 10 μm in zoomed inset image. *site of zoomed image. (b) Quantification of p16^INK4a (red) staining shown in a. grouped by age. *P value <0.05, Student's t-test. Data are mean+s.e.m. Graph contains data from four donors per age group, multiple images (5–17) per donor. (c) IF staining for CD45 (green) in human skin samples grouped by age. Nuclei are DAPI-stained (blue). Scale bar, 100 or 10 μm in zoomed inset image. *site of zoomed image. (d) Quantification of CD45 (green) staining shown in c. grouped by age. *P value <0.05, Student's t-test. Data are mean+s.e.m. Graph contains data from four donors per age group, multiple images (5–17) per patient. (e) IF staining for p16^INK4A (p16, green) and IL-6 (red) on human skin. Nuclei are DAPI-stained (blue). Secondary alone control showed no detectable staining (data not shown). Double-positive staining (yellow) was noted in older individuals (Age 63–73). Scale bar, 100 μm. (f) Quantification of IL-6 IF staining shown in e. Data are the MFI+s.e.m. per × 20 FOV. *P value <0.05, Student's t-test. Four donors per age group, multiple images (4–10) per donor were analysed. (g) IF co-staining for p16^INK4a (red) and CD45 (green) in human skin. Nuclei are DAPI-stained (blue). Panel e is the merged image of the staining shown in a,c. Scale bar, 100 or 10 μm in zoomed inset image. *site of zoomed image. (h) Images from staining shown in e were analysed using the same spatial statistics described in Fig. 1g. In samples from donors aged 63–73, a strong association of p16⁺ and CD45⁺ cells was observed (mean normalization score 2.9, 95% CI 2.4–3.4). Samples from donors aged 29–32 show weak association (mean normalization score 0.8, 95% CI 0.2–1.4). 4 patients per age group. Error bars are s.d. n=27–37. FOV, field of view.