Fig 2: Detection of senescent cell population and SASP cytokines by flow cytometry during early stages of disease progression in Mtb-infected mice.a Schematics of the experimental strategy to measure senescence markers (p21, p16 and SA-βGal activity) and SASP cytokines levels in mice lungs at 10 and 20 days p.i.. (Created in BioRender. Shee, S. (2026) https://BioRender.com/t13mbqd). Young WT B6, B6.Sst1S, and old WT B6 mice were aerosol-infected with 55 CFU of Mtb H37Rv. Mtb H37Rv burden in mice- (b) lungs and (c) spleen are shown at the indicated time points (For WT B6: n = 7-8, B6.Sst1S: n = 8-9, old WT B6: n = 6 from one independent experiment). The data are means ± SEM. Each data point represents a mouse. Statistical analysis was calculated by two-way (b) or one-way (c) ANOVA with Tukey’s multiple comparisons test. (p > 0.05: ns, p < 0.0001: ****). d %p21 + p16+ and (e) %SA-βGal + (CellEvent Senescence green + ) cells out of all live lung (uninfected and Mtb-infected mice) and Spleen (Mtb-infected mice) cells at 10 days p.i. as determined by multicolor flow cytometry. f %p21 + p16+ and (g) %SA-βGal+ cells out of different lung-cell types at 10 days p.i., as determined by multicolor flow cytometry. Gating for p16 + , p21 + , and SA-βGal+ events for each cell type were established using Fluorescence minus one (FMO) controls and reference cells from Mtb-infected WT B6 mice (set at 1%). Squares indicate population not detected. (For uninfected condition: n = 5 mice/ group, For infected condition: WT B6: n = 8, B6.Sst1S: n = 9, old WT B6: n = 6 from one independent experiment) (AMs: Alveolar macrophages, IMs: Interstitial macrophages) (h) Normalized concentration of SASP-cytokines in lung homogenates at 20 dpi and 4 wpi relative to WT B6 mice. The data are means ± SEM. Each data point represents a mouse (For WT B6, B6.Sst1S: n = 12, For old WT B6: n = 11 from one independent experiment). Statistical analysis was calculated by two-way ANOVA with Tukey’s (d–g) or Dunnett’s (h) multiple comparisons test. p-values are indicated in the figure (p > 0.05; ns). Source data are provided as a Source Data file.

Fig 3: Detection of senescence-associated markers during early stages of disease progression in Mtb-infected mice.a Schematics of the experimental strategy to measure senescence markers in vivo at 2- and 4-weeks post-infection. (Created in BioRender. Shee, S. (2026) https://BioRender.com/lwgvtyz) Day1 CFU = 39. Mtb H37Rv burden at indicated time points in WT B6, B6.Sst1S and WT B6 old mice (b) lungs (n = 3 mice/group from one independent experiment) and (c) spleen (n = 5 mice/group from one independent experiment). The data are means ± SEM. p-value was determined by two-way ANOVA with Tukey’s multiple comparisons test and indicated in the figure. Representative H&E-stained and Immunohistochemistry against γH2A.X and p21 (black arrow) of mice lungs at (d) 2 weeks p.i. (turquoise arrow indicates no difference in Mtb burden) and (e) 4 weeks p.i. (c- controlling, nc- noncontrolling lesions) (f) ImageJ quantification of lung pathology (H&E-stained area) in uninfected mice (n = 6 mice/group from one independent experiment) and Mtb H37Rv infected mice lungs (n = 4 mice/group from one independent experiment) at 2 weeks and 4 weeks p.i. Squares indicate necrotic granulomas. g ImageJ quantification of γH2A.X-stained area in uninfected mice and Mtb H37Rv infected mice lungs, and (h) corresponding γH2A.X-IHC intensity at 2 weeks and 4 weeks p.i. (AU. = arbitrary unit; n = 4 mice/group from one independent experiment). The data are means ± SEM. Each data point represents a mouse. p was determined by one-way ANOVA with Dunnett’s multiple comparisons test (f, g: 4 weeks p.i., h) or Kruskal-Wallis with Dunn’s multiple comparison test (uninfected condition, f, g: 2 weeks p.i.) and indicated in the figure (p > 0.05; ns). Source data are provided as a Source Data file.