Fig 2: Phenotypic and molecular characterizations of SHED and DPSCs in senescence. Representative images of SA-ß-gal-positive cells in SHED and DPSCs at (A) P4 and (B) P20. Scale bar, 50 µm. (C) The percentage of SA-ß-gal-positive cells in SHED and DPSCs. (D) Western blot analysis of p53, p21 and p16Ink4a expression in SHED and DPSCs. Reverse transcription-quantitative polymerase chain reaction analysis of p53, p21 and p16Ink4a mRNA expression normalized to GAPDH in (E) SHED and (F) DPSCs at P4 and P20. Data are presented as the mean + standard error of the mean (n=3). *P<0.05, **P<0.01 and ***P<0.001, as indicated. SHED, stem cells from human exfoliated deciduous teeth; DPSCs, dental pulp stem cells; SA-ß-gal, senescence-associated ß-galactosidase; P4/20, passage 4/20.

Fig 3: Chondrocyte-specific temporal Sephs1 knockout exacerbates post-traumatic OA in mice.a Sephs1fl/fl or Sephs1fl/fl; Col2a1-CreERT2 12-week-old mice were injected with TMX five times and subjected to sham operation or DMM surgery. Joint sections were stained with safranin O, fast green, and hematoxylin. The inset in the images is shown as magnified images in the bottom row. b Cartilage destruction, subchondral bone sclerosis, osteophyte formation, and synovial inflammation determined by safranin O/hematoxylin staining and scored (n = 8 for sham-operated WT; n = 5 for sham-operated Sephs1-iCKO; n = 12 for DMM-operated WT; n = 8 for DMM-operated Sephs1-iCKO). c Representative microcomputed tomography (µCT) images of sham- or DMM-operated WT and Sephs1-iCKO mice. d Stress-related selenoproteins (GPX1, SELENOW, and MSRB1), p16INK4a, HMGB1, and e SASPs (MMP13, IL-6, and GROa) were detected by immunohistochemistry in cartilage sections. f Hotplate pain assays in DMM-operated WT and Sephs1-iCKO mice (left panel, n = 12 for WT; n = 8 for Sephs1-iCKO). The percentage of weight placed on the sham- or DMM-operated limb versus the contralateral limb of WT and Sephs1-iCKO mice (right panel, n = 12 for WT; n = 8 for Sephs1-iCKO). Scale bars: a 200 µm, d, e 25 µm. b, f Data represent means ± s.e.m. P values are from Kruskal–Wallis test followed by Mann–Whitney U test (b) or two-tailed t test (f). Cohen’s d effect sizes are provided in Supplementary Table 8. Mankin scores and SBP thickness measurements are provided in Supplementary Figs. 11 and 12.

Fig 4: Separation of PDGFRa+ mesenchymal progenitors and establishment of aging model. (A) Separation of PDGFRa+ mesenchymal progenitors by magnetic beads. (B) Immunofluorescent staining of PDGFRa (scale = 100 µm). (C) Senescence associated ß-galactosidase staining and content of SA-ß-GAL+ cells (%) (scale = 100 µm). (D) Detection of GLB1, p53, and p16 expression through western blotting; GAPDH: internal reference. (E–G) Relative protein-expression levels of GLB1, p53, and p16. (H) Detection of Pim1 expression through western blotting; GAPDH: internal reference. (I) Relative protein-expression level of Pim1. N = 3; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: HIV TAT induces senescence in mouse primary microgliacells (mPMs). (A–B) HIV TAT dose-dependently upregulated the protein levels of senescence markers such as p16 (A) and p21 (B) in mPMs. (C–D) HIV TAT time-dependently upregulated the protein levels of senescence markers such as p16 (C) and p21 (D) in mPMs. ACTB was probed as a protein loading control for all experiments. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn's post hoc test was used to determine the statistical significance of multiple groups. (E) Representative images showing the SA-ß-gal activity (blue color positive cells) in mPMs. Scale bar: 10 µm. (F) Quantification of SA-ß-gal positive mPMs exposed to HIV TAT for 48 h and unexposed control mPMS. (G) Gel image showing telomerase activity in mPMs includes control, HIV TAT (7 nM), heated HIV TAT and positive controls (telomerase containing cell extract and TSP8 primer) using PCR-based TRAP assay. L: ladder. The telomerase signal is visualized as a ladder. Negative controls consisted of heat-inactivated cell extract. The number of PCR cycles was 30. (H) Cell cycle analysis by flowcytometry in mPMs exposed to HIV TAT and unexposed control mPMs. The data are presented as mean ± SEM from six independent experiments. An unpaired Student t-test was used to determine the statistical significance. *, P < 0.05 vs. control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)