Fig. 2: Induction of cellular senescence in fibroblasts by gut bacteria.

a–e, Early passage TIG-3 cells were cultured with tissue culture media containing the indicated bacterial conditioned media or the plain bacterial culture media with (DXR) or without (Mock) doxorubicin at a ratio of 1/30 for 9 days, and then subsequently cultured with plain tissue culture medium for another 3 days. Cell numbers were counted throughout the experiments, and representative photographs of the cells in the indicated culture conditions on day 12 are shown at the top of the panels. These assays were performed in triplicate (both biological and technical replicates) and representative data were shown (a). Cells on day 9 were subjected to RT-qPCR analysis for indicated genes (b), western blotting analysis using antibodies shown right (c), analysis of intracellular ROS levels (d) or to immunofluorescence staining for markers of DNA damage (γ-H2AX (green) and pST/Q (red)) and DNA staining with 4′, 6-diamidino-2-phenylindole (DAPI) (blue) (e). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining (e). The assay was performed three times and representative data is shown (c). f TIG-3 cells were cultured with or without (Mock) the indicated bacterial culture supernatants for 9 days, and then subjected to annexin V and Propidium iodide (PI) staining analyses. Actinomycin D treated cells were used as a positive control for apoptotic cells, and doxorubicin (DXR) treated cells were used as a positive control for senescent cells. The histogram shows the percentage of cells that were positive for Annexin V (green bar), PI (red bar) or both (blue bar), respectively. Double positive cells represent apoptotic cells. For all bar graphs, error bars indicate mean ± standard deviation (s.d.) with three biologically independent replicates. Statistical significance was determined with one-way ANOVA followed by Tukey’s test (b), (d), (e) or two-tailed Dunnett’s test for comparing with mock (f). P values < 0.05 were considered significant. Source data are provided as a Source Data file.