Fig. 1.

Levels of secreted EVs increase with cell senescence. (A) Senescence was monitored by assessing the senescence-associated (SA)-β-gal activity (blue staining) of WI-38 cells and confirmed in three different senescence paradigms, while proliferating cells (P) do not show staining. WI-38 human diploid fibroblasts were either left untreated (Proliferating, P) or were rendered senescent by exposure to 10 Gy ionizing radiation (IR) followed by incubation for 8 d, by treatment with 50 μM etoposide every 72 h and harvested at day 6 (ETO) or reached replicative senescence (RS) by proliferation to exhaustion, from population doubling number (PDL) 20 (P fibroblasts) to senescence at ~PDL52. (B) The levels of the senescence marker protein p53 (TP53) in each of the four fibroblast populations were monitored by western blot analysis; the levels of the housekeeping control protein GAPDH were assessed to monitor differences in loading across the four groups. (C) Representative transmission electron microscopy (TEM) images showing the morphology of S-EVs from fibroblasts rendered RS as described in A. See also SI Appendix, Fig. S1B. (D) Size and concentration (EVs per mL) of EVs collected from the populations described in A and measured by Nanosight NS300 Nanoparticle tracking analysis (NTA, Methods). (E) The numbers of EVs per cell were calculated based on measurements taken over 48 h in the fibroblast populations described in A. Data in E are the mean ± SEM from three biological replicates; ***P ≤ 0.001. Data were analyzed by one-way ANOVA.