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. Author manuscript; available in PMC: 2014 Feb 1.
Published in final edited form as: Nat Cell Biol. 2013 Jun 16;15(8):978–990. doi: 10.1038/ncb2784

Figure 2. Paracrine senescence is a stable arrest mediated by soluble factors.

Figure 2

(a) Quantification of the percentage of IMR90 ER:RAS (centre) and IMR90 Cherry cells (right) incorporating BrdU during the course of 7 days after activation with 4OHT in co-cultures. Data is a representative experiment. The source data for 2 independent experiments are provided in Supplementary Table S8. (b) Kinetics of the production of secreted factors during OIS. Conditioned media from IMR90 ER:RAS cells was collected and used to detect CXCL1 (left) or IL-8 (right) levels by ELISA. Data is a representative experiment. The source data for 2 independent experiments are provided in Supplementary Table S8. (c) Paracrine senescence is a stable arrest. Cells were co-cultured separated by transwells. After a week IMR90 cells in the bottom were split, seeded and cultured alone for 14 additional days. CV (centre) and SA-β-Gal staining (right) is shown. Scale bar, 50 μm. (d) Conditioned media from senescent cells induces paracrine senescence. Diagram explaining the CM experiments (left). CM was collected from IMR90 vector or IMR90 ER:RAS cells grown 7 days in the presence 200 nM 4OHT. The effect of CM on IMR90 cells was evaluated by BrdU staining (right). Data is a representative experiment of n=2 independent experiments for days 1-3 and mean +/− s.d. of n= 3 independent experiments for day 4. The source data for 2 independent experiments and statistics for day 4 (Student’s t-test) are provided in Supplementary Table S8. (e) Fibroblast undergoing OIS induce growth arrest on normal neighboring fibroblasts. IMR90 ER:RAS cells were seeded in ‘clusters’ surrounded of normal IMR90-Cherry cells with the help of cloning cylinders (left). Cells were grown in the presence or absence of 200 nM 4OHT for 7 days. Percentage of BrdU positive cells was calculated in consecutive fields in the external (IMR90-Cherry) and internal region (IMR90-ER:RAS). Data is mean +/− s.d. of n= 3 optical fields. (f) An experiment testing serial transfer of senescence by conditioned media showed a limited arrest with absence of SA-β-Gal positive cells in ‘tertiary’ cells. Data is representative of n=2 independent experiments. Scale bar, 50 μm.