Fig 1: Isolated CD24+ bone stromal cells exhibit functional characteristics of senescence and impaired osteogenesis.a Gating strategy for FACS isolation of CD24+ and CD24− skeletal stromal cells from Lin-depleted bone/marrow cell suspensions; b Gating strategy and c quantification of cell cycle analysis of CD24+/− cells (n = 7 mice); d Outline of in vitro senescence phenotyping of CD24+/− cells; I Brightfield images and quantification of colony formation efficiency (CFE) assay and SA-β-gal staining of CD24- and CD24+ cells after 7 or 14 days in culture, respectively (n = 3 mice); f Outline of osteoblast differentiation assays of CD24+/− cells; g CD24- and + cell monolayers stained with either alkaline phosphatase (ALP) or Alizarin Red after 14 and 21 days in culture, respectively. Accompanying high magnification images are 20X (n = 3 mice); h Schematic of in vitro etoposide-induced senescence of BMSCs; I MRNA levels of Cdkn2aINK (p16), Cdkn1a (p21), and Cd24a (CD24) from BMSCs treated with vehicle (DMSO) or etoposide (n = 3 mice). Schematic in (h) was generated using BioRender. Scale bars represent 750 μm. Bars show mean ± SD (c, e). Two-sided unpaired t-test or Mann–Whitney test as appropriate; (i) Multiple two-sided t tests with Holm–Sidak Correction. Source data are provided as a Source Data file.
Fig 2: CD24high osteolineage cells represent inflammatory senescent cells in old mice targeted by genetic senolytic clearance.a t-SNE visualization and FlowSOM clustering of n = 80,000 CD45-Lin- bone and marrow cells (n = 40 INK-ATTAC mice [n = 15 young, n = 12 old + vehicle, n = 13 old + AP] – 2,000 cells sampled per mouse) analyzed by CyTOF. Cells are colored by clustered population (see Table 1 for defining markers); b Heatmap representation of the 11 cell clusters and protein expression of identification markers; c Log2 fold-Change mean expression of p16 or BCL-2 in each cluster with age. d Log2 fold-change of %p16KB cells in each cluster across aging; e Schematic of p16+ senescent cell clearance in 24-month-old AP-treated INK-ATTAC mice; f t-SNE plots of FlowSOM clusters of bone/bone marrow cells from old vehicle- or AP-treated INK-ATTAC mice. Black arrows indicate cleared clusters while green arrows indicate increased cluster abundance; g Quantification of cluster abundance changes between vehicle- and AP-treated mice (Log2-fold change to vehicle); h CD24 expression feature plots in samples from vehicle- or AP-treated mice; i Feature plots of p16 and FLAG (INK-ATTAC transgene) protein expression; j Heatmap and k, l volcano plots for statistical comparisons of senescence marker expression between cleared clusters in vehicle-treated old mice. Schematic in (e) was generated using BioRender. Box plots show median and interquartile ranges with error bars representing minimum and maximum values. (c, d, g) Two-sided Mann–Whitney test or unpaired t test, as appropriate. k, l Multiple two-sided t tests with Holm-Sidak Correction. Source data are provided as a Source Data file.
Fig 3: CD24 osteolineage cells display an age-related senescence expression profile.a Schematic of CITRUS analysis workflow; b Aging markers upregulated in the highest number of CITRUS clusters with age (FDR < 5%); c Senescence/SASP marker expression changes across aging overlaid on CITRUS plot (each circle is a cluster, grouped by cluster families dotted lines; see Supplementary Fig. 6A for identity marker expression). Red clusters indicate upregulated p16 expression, while colored overlays indicate upregulation in the corresponding SASP marker. All markers converge in CD24+/Runx2+/Osterix+ clusters, indicating that this population exhibits the most widespread age-related increase in SASP marker expression in bone/bone marrow. Box plot below demonstrates the upregulation of %p16KB cells with age in this population. Red arrows point to d quantified median expression of various SASP factors in the CD24+/Runx2+/Osterix+ clusters with age (mean ± SD) and e overlay of the CD24+/Runx2+/Osterix+ cluster on original t-SNE plot with FlowSOM clusters labeled (c, d) n = 15 young and n = 12 old biologically independent animals; f UMAP visualization by scRNA-seq of n = 3,362 clustered Lin-CD45- cells from the digested bone and marrow of n = 3 24-month untreated male INK-ATTAC mice; CAR, Cxcl12-abundant reticular; g Density plots of CD24 osteolineage markers and anti-apoptotic factors (Bcl2, Bcl2l1, Bcl2l2). Schematic in (a) was generated using BioRender. Box plots show median and interquartile range with error bars representing minimum and maximum values. c Two-sided Mann–Whitney test. d Multiple two-sided t-tests with Holm-Sidak Correction. Source data are provided as a Source Data file.
Fig 4: Validation of antibodies and detection of senescent cells by CyTOF.a Experimental workflow of single mouse protein expression in U2OS cells for the testing of CyTOF antibodies; b qPCR analysis confirming upregulated mouse p16Ink4a mRNA (Cdkn2a) after expression vector transfection (n = 3 independently run experiments; mean ± SD); c CyTOF dot plots of expression samples testing different p16 antibodies, demonstrating outcomes of both failed (antibody #1) and successful (antibody #2) tests; d Schematic of testing senescence-specific CyTOF antibodies using etoposide-treated mouse BMSCs with qPCR confirmation of upregulated p16Ink4a (Cdkn2aINK) and p21Cip1 (Cdkn1a) transcripts (n = 3 independently run experiments; mean ± SD); e CyTOF plots of p16 and p21 protein expression, demonstrating increased percent-positive cells with etoposide treatment; f Fold-change of percent-positive values for each of the senescence panel markers in etoposide-treated cells, where dotted line represents values from vehicle. g Schematic of bone and marrow mesenchymal cell isolation and CyTOF workflow; h Gating strategy for p16+ cells using similarly processed cells from p16-null mice as a negative control (i) Quantification of %p16+ and (j) %p21+ cells in young and old mice (n = 15 young and n = 12 old biologically independent animals). Schematics in (a, d, g) were generated using BioRender. Box plots show median and interquartile range with error bars representing minimum and maximum values. (d) Multiple two-sided t tests with Holm-Sidak Correction. (i, j) Two-sided Mann-Whitney test. Source data are provided as a Source Data file.
Fig 5: BCL-2 expression defines p16+ cells with senescent characteristics.a Schematic of multidimensional p16+ senescent cell analysis workflow; b UMAP visualization and FlowSOM clustering of pooled p16+ cells from young (n = 15) and old (n = 12) mice with c bar graphs indicating percent-of-whole cluster abundance changes. d FlowSOM clusters ranked by descending Ki67 mean expression plotted against p16 mean expression (Log2 Fold-Change). e Gating strategy for p16 + BCL2+ cells and f quantification of %Ki67+ and Ki67 mean expression alongside p16- and total p16+ cells. g Volcano plots of age-related changes in mean senescence marker expression within p16+ cells and quantification of % BCL-2+ cells and mean BCL-2 expression within all p16+ cells with age; h Gating strategy for “p16KB” cells; i Heatmap representation of protein expression between p16KB cells and non-senescent p16- cells, with asterisks indicating significance (*p < 0.05, **p < 0.01). Columns within each population represent a biological replicate (n = 12 Old). j Quantification of p16KB cells with age and numerical comparison to total p16+ cells (n = 15 young and n = 12 old biologically independent animals). Schematic in (a) was generated using BioRender. Box plots show median and interquartile range with error bars representing minimum and maximum values. (f, g [volcano plot]) Multiple two-sided t tests with Holm-Sidak Correction. (g, i, j) Two-sided Mann–Whitney test. Source data are provided as a Source Data file.
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