Figure 1: Senescent stromal cells induce local inflammation.

(a) Senescence activation in FASST or littermate mice. Senescence-associated β-galactosidase (SA-βgal) staining (blue) of skin (arrows). Scale bar, 100 or 10 μm in zoomed images. * site of zoomed image. Representative images. (b) Quantification of SA-βgal⁺ cells per × 20 field of view (FOV). *P-value<0.05 by Student’s t-test. Error bars represent s.e.m. Representative experiment. n=10. (c) RNA-seq shows expression of multiple cytokines and chemokines significantly increased in senescent MSFs (FDR<0.05, edgeR). Graph is log fold change (logFC) expression in Sen compared with Non-Sen MSFs. n=3. (d) CD45 IHC staining of skin from FASST or littermate mice. Arrows indicate CD45⁺ cells. Scale bar, 100 μm. Representative images. (e) Quantification of CD45⁺ cells per × 20 FOV. *P value<0.05 by Student’s t-test. Error bars represent s.e.m. Representative experiment. n=9. (f) Schematic of transgenic expression cassette (top). Below, IF co-staining for transgenic GFP expression (green) and CD45⁺ cells (red). Nuclei stained with DAPI (blue). Scale bar, 100 or 10 μm in zoomed image. * site of zoomed images (right). Representative image. (g) Spatial analysis of CD45⁺ leukocytes and GFP⁺ stromal cells. Data show median distance between CD45⁺ leukocytes and GFP⁺ stromal cells (green) is significantly smaller than chance (mean normalized association score 3.5, 95% confidence interval (CI) 2.4–4.6). In the absence of senescent cells (littermate control), the observed distance distribution of immune cells to the epithelial layer (orange) is not significantly different from random (Epithelium-CD45) (mean normalized association score=1.0, 95% CI −0.2 to 2.2). Immune cell-immune cell (CD45-CD45) distances (red) are not significantly different from random (mean association statistic=0.2, 95% CI −0.5 to 0.9). Error bars represent s.d. n=6–9. (h) Flow cytometry analysis of FASST and littermate control skin. Data represents CD45⁺, CD45⁺CD11b⁺ or CD45⁺CD3⁺ populations as a percentage of live cells per mouse skin. *P value <0.05 by Student’s t-test. Data are presented as mean %+s.e.m. shown as error bars. n=3. FDR, false discovery rate.