Figure 2: Senescent fibroblasts drive inflammation.

(a) MSFs isolated from FASST mice show activation of the GFP transgene following addition of 10 μM TAM. Flow cytometry plots with the percent of cells expressing GFP (lower right corner) in non-senescent (Non-Sen) and senescent (Sen) MSFs. Representative plot. n=5. (b) Senescence-associated β-galactosidase (SA-βgal) staining (blue) on MSFs from FASST mice treated with 10 μM TAM (Sen) or vehicle (Non-Sen). Scale bar, 100 μm. Representative images. (c) Quantification of SA-βgal staining. Data are presented as mean percent positive cells+s.e.m. * indicates P value <0.05 by Student’s t-test. n=3. (d) Quantification of secreted IL-6 protein from Sen or Non-Sen MSFs. Data represents the mean concentration of IL-6 in pg ml⁻¹+s.e.m. from medium conditioned for 24 h. Protein concentration was subsequently measured using ELISA. *P value <0.05 by Student’s t-test. n=3. (e) IL-6 mRNA expression from MSFs (Sen or Non-Sen) measured by qRT–PCR. Graph represents mean IL-6 mRNA expression in senescent versus non-senescent MSFs. * indicates P value<0.05 by Student’s t-test. Error bars are s.e.m. Representative experiment. n=10. (f) H&E staining of adjacent skin, matrigel alone, non-senescent (Non-Sen) and senescent (Sen) subcutaneous isografts. Scale bar, 100 μm. Representative images. * indicates site of zoomed images (right). Scale bar, 20 μm in the zoomed images. n=4–6. (g) IF for CD11b⁺ (red) cells present in Non-Sen and Sen subcutaneous isografts. Scale bar, 100 μm. Representative images. n=3. (h) IF for Gr-1⁺ (red) cells present in Non-Sen and Sen subcutaneous isografts. Scale bar, 100 μM. Representative images. n=3. (i) Quantification of CD11b⁺ cells shown in g. *indicates P value <0.05 by Student’s t-test. Data are presented as the mean+s.e.m. of CD11b⁺ cells per × 20 field of view (FOV). Representative experiment. n=3. (j) Quantification of Gr-1⁺ cells shown in h. * indicates P value <0.05 by Student’s t-test. Data are presented as the mean+s.e.m. of Gr-1⁺ cells per × 20 FOV. Representative experiment. n=3. H&E, haematoxylin and eosin; qRT–PCR, quantitative PCR with reverse transcription.