Research Paper Volume 4, Issue 12 pp 932—951

Figure 2. Medium from various types of senescent cells induces senescence in bystander cells. (A) Senescence-associated β-galactosidase detection and (B) immunofluorescence detection of PML nuclear bodies (PML NBs) in BJ cells treated with senescent medium from drug-induced (DSM), oncogene-induced (OSM) or replicative (RSM) senescent BJ cells for 20 days. BJ cells treated with medium from non-senescent BJ cells (CM) were used as a control. Bar 100 μm for (A), 15 μm for (B). (C) Statistical analysis of BrdU incorporation in BJ cells treated for 4 days with conditioned medium from replicative senescent cells (RSM). BJ cells treated 4 days with medium from non-senescent cells (CM) were used as a control. (D) Immunoblot detection of PML, total STAT1, STAT3 and STAT5, STAT1 phosphorylated on tyrosine 701 (STAT pY701), STAT3 phosphorylated on tyrosine 705 and serine 727 (STAT3 p705 and pS727) and STAT5 phosphorylated on tyrosine 694 (STAT5 pY694). (E) Plasminogen activator inhibitor (PAI) mRNA levels quantified by real time qRT-PCR in BJ cells treated with senescent medium from drug-induced (DSM), oncogene-induced (OSM) or replicative (RSM) senescent BJ cells for 20 days. BJ cells treated with medium from non-senescent BJ cells (CM) were used as a control. The mRNA values represent average of two independent experiments and are shown as a fold induction relative to control BJ cells (CM); error bars represent standard error. β-actin was used as a reference gene. (F) PAI mRNA levels quantified by real time qRT-PCR in drug-induced (DIS), oncogene-induced (OIS) and replicative senescent (RS) BJ cells. The mRNA values represent average of two independent experiments and are shown as a fold induction relative to control BJ cells (ctrl) or BJ cells transfected with empty vector (empty); error bars represent standard error. β-actin was used as a reference gene.