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Conditioned medium from apoptotic cardiomyocytes stimulated EPC migration, while conditioned medium from senescent cardiomyocytes did not produce any effect (Fig. 5).

In order to obtain conditioned medium from senescent or apoptotic H9c2, we treated the cells for 3 hours with 0.1 or 1 µM doxorubicin, respectively, in growth factor-free DMEM plus 0.1% BSA, then the H9c2 were grown in fresh medium (growth factor-free DMEM plus 0.1% BSA) for 24 hours [9].

Taken together, these results show that conditioned medium from senescent cells decreases FLIPL levels via MYC-mediated repression and confers sensitivity to otherwise TRAIL-resistant pretransformed cells.

Conditioned medium from senescent fibroblasts alone was not sufficient to increase the foci formation rate (Fig. 1E,F) or the steady-state foci frequencies (Fig. S3).

Conditioned medium was collected after filtration (0.20-μm Minisart High-Flow syringe filter; Sartorius, Goettingen, Germany) of 2-day-old medium from senescent cells or replicating counterparts.

After validating the tumor specificity of TRAIL action in the stepwise human tumorigenesis model (Hahn et al., 1999), we incubated the cells at different stages of transformation with conditioned medium from senescent cells (CMS) for 20 h and treated them with TRAIL.

Conditioned medium from the senescent fibroblasts recapitulated these findings, indicating that a large component of the growth-promoting effect was due to paracrine-acting factors.

Indeed, probing of control and bRS cells (i.e. BJ cells treated with medium from replicative senescent cells) with 2',7'-dichlorofluorescein indicated elevated levels of ROS in bRS cells (Fig. 3C).

The increased activation of Arg-II and p38 was associated with enhanced secretion of the inflammatory cytokines IL-6 and IL-8 measured in the conditioned medium from the senescent cells as compared to that from the young cells (Fig. 1C).

Importantly, however, the exposure of the U2OS tumor cell line to conditioned medium from drug-induced senescent U2OS cells did result into development of bystander senescence with expressed hallmarks of senescence, analogous to the scenario seen in normal BJ cells (Suppl. Fig. 2E-H).

We also found that conditioned medium from doxorubicin-induced senescent cardiomyocytes does not attract untreated EPCs, unlike conditioned medium from apoptotic cardiomyocytes which has a strong chemoattractant capacity.