Research Paper Volume 4, Issue 12 pp 932—951
IL1- and TGFβ-Nox4 signaling, oxidative stress and DNA damage response are shared features of replicative, oncogene-induced, and drug-induced paracrine ‘Bystander senescence’
Figure 4. NFκB and TGFβ induce DNA damage in bystander cells. (A) Detection of cytokines in medium of parental senescent cells using FACS beads assay. The values are shown as a fold induction relative to non-treated BJ cells (ctrl). (B) Immunoblot detection of serine 139 phosphorylated H2AX (γH2AX) and STAT3 phosphorylated on tyrosine 705 (STAT3 pS705) in BJ cells treated with control (CM) or replicative senescent medium (RSM) in presence or absence of IL6 neutralizing antibody (2 μg/ml). (C) Immunoblot detection of γH2AX and STAT3 pS705 in BJ cells treated with CM or RSM in presence or absence of JAK kinase family specific inhibitor (JAKi; 0.25 μM). (D) Immunofluorescence detection of the p65 subunit of NFκB in BJ cells treated with senescent medium from drug-induced (DSM), oncogene-induced (OSM) or replicative (RSM) senescent BJ cells for 4 days. BJ cells treated with medium from non-senescent BJ cells (CM) were used as a control. Bar 15μm. (E) Immunoblot and (F) indirect immunofluorescence detection of γH2AX in BJ cells treated with CM or RSM in presence or absence of IL1 receptor antagonist (IL1R ant.; 25 μM). Bar 15μm. (G) Immunoblot detection of γH2AX, NEMO and SMAD2 phosphorylated on serine 465/467 (SMAD2 pS465/467) and (H) immunofluorescence detection of γH2AX foci in BJ cells treated with CM or RSM in presence or absence of TGFβ receptor inhibitor (TBRi; 10 μM), NEMO siRNA or combination of both. Cells without transfection with siNEMO were transfected with control siRNA. Bar 15μm. (I) Detection of ROS production by 2',7'-dichlorofluorescein (DCF) staining in BJ cells treated with CM or RSM in presence or absence of TGFβ receptor inhibitor (TBRi; 10 μM). Bar 15μm. All these experiments (except experiment A and C) were measured in BJ cells treated 4 days with conditioned medium from replicative senescent cells (RSM; diluted 1:1 with fresh medium). BJ cells treated 4 days with medium from non-senescent cells (CM; diluted 1:1 with fresh medium) were used as a control.