The induction of the fibroblast extracellular senescence metabolome is a dynamic process

Cellular senescence is often associated with irreparable DNA double strand breaks (IrrDSBs) which accumulate with chronological age (IrrDSBsen). The removal of senescent cells ameliorates several age-related diseases in mice but the translation of these findings into a clinical setting would be aided by the characterisation of non-invasive biomarkers of senescent cells. Several serum metabolites are independent indicators of chronological age and some of these accumulate outside senescent fibroblasts independently of cell cycle arrest, repairable DNA breaks and cell size (the extracellular senescence metabolome, or ESM). The post-mitotic phase of senescence is dynamic, making the detection of senescent cells in vivo difficult. An unbiased metabolomic screen of the IrrDSBsen fibroblast ESM also showed differences in the times of initiation and maintenance of different metabolites but generally the ESM altered progressively over the 20 day study period unlike the reported transcriptional profiles. This more detailed analysis of IrrDSBsen identified several new ESM metabolites that are associated with chronological ageing. Targeted analysis of citrate confirmed the dynamic nature of this metabolite in two cell lines and revealed its independence from the senescence effector p16INK4A. These data will aid our understanding of metabolic signatures of ageing and their relationship to cellular senescence and IrrDSBs.

Representative western blot showing MCM7 protein levels in IMR90 cells treated with 0Gy, 0.5Gy and 20Gy on days 5, 10 and 20 after treatment. Blots were performed separately on lysates from three independent experiments. HeLa is a positive control.
White space between rows of bands depicts where the membrane has been stripped and re-probed. The exposure times may be different for each section of membrane.
Uncropped images of films used to make this figure can be seen in supplementary figures S18A and S18B.
Representative western blot showing MCM7 protein levels in NHOF-1 cells treated with 0Gy, 0.5Gy and 20Gy on days 5, 10 and 20 after treatment. Blots were performed separately on lysates from three independent experiments. HeLa is a positive control.
White space between rows of bands depicts where the membrane has been stripped and re-probed. The exposure times may be different for each section of membrane.
Uncropped images of films used to make this figure can be seen in supplementary figures S18C and S18D.
A ImageJ nuclear overlays (magenta dotted lines) on 53BP1 foci (green) in NHOF1 after 0, 0.5 or 20Gy ionizing radiation on 5, 10 and 20 day time points. Scale bar represents 50µm. Staining and image acquisition protocols are described in supplementary method 1 B shows the mean score for each group across three repeats. Counts were obtained using ImageJ, by the method described in supplementary method 3. Error bars represent standard deviation from the mean. NS = not significant ***p<0.001, 2 way ANOVA with Tukey's post hoc analysis.
A ImageJ nuclear overlays (magenta dotted lines) on 53BP1 foci (green) in IMR90 after 0, 0.5 or 20Gy ionizing radiation on 5, 10 and 20 day time points. Scale bar represents 50µm. Staining and image acquisition protocols are described in supplementary method 1 B shows the mean score for each group across three repeats. Counts were obtained using ImageJ, by the method described in supplementary method 3. Error bars represent standard deviation from the mean, n=3 in 5 and 10 day groups, n=2 in day 20 group due to damage on the slide. NS = not significant with 1 way ANOVA.
Supplementary Figure  Representative western blots of MCM7 protein levels in PEsen, growing (Gr), serum starved (LS) and confluent (Con) controls with β-actin loading control, in IMR90 (A) and NHOF-1 (B). Blots were performed separately on lysates from three independent experiments. White space between rows of bands depicts where the membrane has been stripped and re-probed. The exposure times may be different for each section of membrane. Uncropped images of films used to make this figure can be seen in supplementary figures S18G and S18H.
Supplementary Figure S16. p16 INK4A protein level in PEsen IMR90 and growth arrest controls.
Representative western blot of p16 INK4A protein levels in PEsen IMR90 cells as well as growing (Gr), serum starved (LS) and confluent (Con) controls, with β-actin loading control. White space between rows of bands depicts where the membrane has been cut for incubation with different primary antibodies. The exposure times may be different for each section of membrane. Uncropped images of films used to make this figure can be seen in supplementary figures S18H and S18I.
Representative western blot of p16 INK4A protein levels in PEsen NHOF-1 cells as well as growing (Gr), serum starved (LS) and confluent (Con) controls, with β -actin loading control. Blots were performed separately on lysates from three independent experiments. White space between rows of bands depicts where the membrane has been cut for incubation with different primary antibodies. The exposure times may be different for each section of membrane. Uncropped images of films used to make this figure can be seen in supplementary figures S18H and S18I.
Supplementary figure S18A: white box shows area of membrane and where membrane was cut prior to antibody incubations. Yellow box shows the MCM7 bands that are used in figure S8 Supplementary figure S18B: white box shows area of membrane and where membrane was cut prior to antibody incubations. Yellow box shows the beta actin bands that are used in figure S8 Supplementary figure S18C: white box shows area of membrane and where membrane was cut prior to antibody incubations. Yellow box shows the MCM7 bands that are used in figure S9.
Supplementary figure S18Di: white box shows area of membrane and where membrane was cut prior to antibody incubations. The top yellow box shows the beta actin bands that are used in figure S9 and S13, the bottom yellow box shows the p16 INK4a bands that are used in figure S13, after 5 minutes exposure of the film to the membrane.
Supplementary figure S18Dii: the same blots shown in SF1a but at an exposure time of 20 seconds.
Supplementary figure S18E: white box shows area of membrane and where membrane was cut prior to antibody incubations. Yellow box shows the p16 INK4a bands that are used in figure S12 Supplementary figure S18F: white box shows area of membrane and where membrane was cut prior to antibody incubations. Yellow box shows the beta actin bands that are used in figure S12 Supplementary figure S18G: white box shows area of membrane and where membrane was cut prior to antibody incubations. Top yellow box shows the MCM7 bands that are used in figure  S15B, bottom yellow box shows the MCM7 bands that are used in figure S15A.
Supplementary figure S18H: white box shows area of membrane and where membrane was cut prior to antibody incubations. Top yellow box shows the beta actin bands that are used in figure  S15B, bottom yellow box shows the beta actin bands that are used in Figure S15A.
Supplementary figure S18I: white box shows area of membrane and where membrane was cut prior to antibody incubations. Top yellow box shows the p16 INK4A bands that are used in figure  S16, bottom yellow box shows the p16 INK4A bands that are used in Figure S17. Supplementary