Fig. 2.

Role of SASP and SAMD for the bystander effect. A: Cartoon model representing the in vitro model system designed to analyse the senescent bystander effect. Three different modes of cellular senescence were employed in the inducer cells: replicative senescence in MRC5 (Rep Sen), oncogene induced senescence via RAS in IMR90 (OIS), and X-ray irradiation induced senescence (10 days post 20 Gy) of either MRC5 or IMR90 (IR). In each case, young proliferating MRC5 cells stably expressing mcherry-53BP1 were used as bystander cells, cultured in a 1:1 ratio, and mcherry-53BP1 foci were counted in bystander cells to determine the level of DNA damage. B: Representative images of co-cultured bystander cells (mCherry-53BP1 expressing MRC5) in the presence of inducer cells (MRC5 top, IMR90 bottom). The mode of senescence for each inducer type is shown in the image overlays. Dashed outlines highlight inducer cell nuclei. Blue represents DAPI staining, red mCherry-53BP1. Note cytosolic autofluorescence present in senescent inducers. Scale bar = 10 μm. C: All modes of senescence induce a bystander response. Co-cultures were grown for 7 days with either young proliferating control cells (Prolif.) or inducer cells in the indicated senescence mode. 53BP1 foci frequencies in the bystander cells are shown. Significant differences (* P < 0.05) in comparison to respective controls are indicated, Kruskal-Wallis ANOVA on Ranks with Holm-Sidak post hoc test for pairwise comparison between groups. Neither cell strain nor senescence mode had a differential impact on the bystander effect (Two-way ANOVA test). Data plotted are mean +/− SD from 100 to 150 cells per group from 3 independent replicates. D, E: ROS and NF-κB contribute to the bystander response. Proliferating control (Prolif or −RAS) or senescent inducer cells as indicated were co-cultured with proliferating MRC5:53BP1 bystander cells in the presence of either Catalase (Antiox., 100U/ml), NF-κB inhibitor Bay11-7082 (1 μM) or carrier (DMSO). Cultures were co-cultured for either 6 days (D) or 21 days (E). 53BP1 foci frequencies in the bystander cells are shown. CAT significantly inhibited the bystander effect in all experiments, while Bay11-7082 reduced the bystander effect from MRC5-RS only after prolonged co-culture. Data plotted are mean +/− SD from 180 to 250 cells per group from 2 independent replicates. Kruskal-Wallis ANOVA on Ranks with Holm-Sidak post hoc test for pairwise comparison between groups, asterisks show P < 0.05. F: ΔIκBα expression inhibits the bystander effect from replicatively senescent MRC5. Young (Prolif) or replicatively senescent (RS) MRC5 fibroblast stably transduced with either mCherry or ΔIκBα we co-cultured with MRC5:53BP1 bystander cells. Differences between 53BP1 foci frequencies in bystander cells to those in proliferating cells alone are shown. Experiments were performed in triplicate and between 60 and 120 cells were analysed per treatment and replicate. Data are mean ± SE (n = 3-5), with approximately 100 cells per experiment. G: TNFα pre-stimulation enhances the bystander effect, and CAT abrogates it. Senescent inducer cells were stimulated with TNFα (TNF) or not (UNSTIM) before co-culture with reporter cells for 21 days. CAT (ANTIOX) was added during co-culture. Differences between 53BP1 foci frequencies in bystander cells to those in proliferating cells alone are shown. Data are mean ± SE (n = 3-5), with approximately 100 cells per experiment. H: Inhibitors of NF-κB, mTORC1 and p38MAPK equally suppress the bystander effect. Replicatively senescent MRC5 fibroblasts were co-cultured with reporter cells for 21 days with either DMSO as carrier, Bay11-7082 (BAY), the mTORC1 inhibitor torin1 (TOR) or the p38MAPK inhibitor UR-13756. Differences between 53BP1 foci frequencies in bystander cells to those in proliferating cells alone are shown. Data are mean ± SE (n = 3-5), with approximately 100 cells per experiment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)