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 3. Increase of DNA damage, ROS production and mitochondrial membrane potential in cells treated with senescence-conditioned medium. (A) Immunoblot (time course from 0 to 6 days) and (B) indirect immunofluorescence (day 4) detection of γH2AX in BJ cells treated with conditioned control (CM) and replicative senescent cell medium (RSM); GAPDH was used as a loading control. Bar 15 μm. (C) Immunofluorescence detection of ROS production by 2',7'-dichlorofluorescein (DCF) staining and (D) detection of mitochondrial potential by tetramethylrhodamine ethyl ester (TMRE) in BJ cells treated with conditioned control (CM) and replicative senescent cell medium (RSM). Bar 15 μm. (E) Immunoblot detection of γH2AX in BJ cells treated with normal or senescent medium in presence or absence of N-acetylcysteine. All these experiments except (A) 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.