Fig 2: Damaged endothelium, intraplaque hemorrhage and neovascularization. (A) Immunofluorescence staining of the brachial artery in Fbn1C1039G+/-LDLR-/- mice and LDLR-/- mice after 24 weeks on a HFD. (B) Immunofluorescence staining of the aortic sinus in Fbn1C1039G+/-LDLR-/- mice and LDLR-/- mice after 24 weeks on a HFD. (C) Immunofluorescence staining quantification of the CD31-positive area (% of plaque) in Fbn1C1039G+/-LDLR-/- mice and LDLR-/- mice after 24 weeks on a HFD. (D) Immunofluorescence staining statistics of the TER119-positive area (% of plaque) in Fbn1C1039G+/-LDLR-/- mice and LDLR-/- mice after 24 weeks on a HFD. (E) Quantification of the CD31-positive area (% of media and adventitia) in Fbn1C1039G+/-LDLR-/- mice and LDLR-/- mice after 24 weeks on a HFD (n = 5 in Fbn1C1039G+/-LDLR-/- mice, n = 3 in LDLR-/- mice), ***p < 0.001. Magnification: ×100. TER119 shows red blood cells, CD31 shows endothelial cells, and DAPI shows nuclei. The arrow indicates the endothelial barrier, the triangle indicates neovascularization, and *indicates intraplaque hemorrhage. P, plaque; L, lumen.

Fig 3: TTLL4 modifies NAP1 in RBCs. (A) Immunoblot showing 60-kDa glutamylated protein in whole red blood lysate of wild-type and Ttll4-KO mice. mAb GT335 was used to detect glutamylation. (B) Immunoblots (IB) after immunoprecipitation (IP) of 60-kDa glutamylated protein from RBC lysate performed with GT335 antibody. (C) Proteins immunoprecipitated with anti-NAP1 antibody. (D) Confocal microscopy images after immunohistochemistry showing NAP1 (magenta) in RBCs. TER-119 (green) is the erythroid marker. Scale bar, 5 µm. (E) Immunoblotting analysis of levels of glutamylated NAP1 in cytosolic and membrane fraction of wild-type and Ttll4-KO RBCs. Carbonic anhydrase1 (CA1) and glycophorin A (GPA) were used as cytosolic and membrane markers, respectively. (F) Quantification of glutamylation levels of NAP1 as described in E. Data are shown as mean ± SEM (three mice per group; *p < 0.05). Mice 8–12 mo of age regardless of the sex were used for each experiment.

Fig 4: Age-related changes in blood and BM populations and altered lineage bias in old MPPs.a, Overlap between cytokines upregulated in old BM fluids and published SASP profile40. b, Representative SA-b-gal staining in control irradiated mouse embryonic fibroblasts (MEF) and isolated young and old MSC-S and OBC. Scale bar, 20 µm. c, Absolute expression of Il1b in different mature hematopoietic cell types and unfractionated (CD45-/Ter119-) endosteal and central marrow stroma from pooled young and old samples. Results are expressed as -log(dCt) relative to gapdh. d, Complete blood count (CBC) parameters in young and old mice. Results are from 7 independent cohorts. WBC, white blood cell; My, myeloid (neutrophil + basophil + eosinophil); Ly, lymphocyte; RBC, red blood cell; Pt, platelet. e, Cellularity and quantification of mature populations in young and old BM. Results are from 3 independent cohorts. Gr, Mac-1+/Gr-1+ granulocyte; B cell, B220+ B cell; T cell, CD3+ T cell. f, Quantification of progenitor populations in young and old BM. Results are from 3 independent cohorts. CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; MEP, megakaryocyte-erythrocyte progenitor; CLP, common lymphoid progenitor; CFU-E, erythroid colony-forming unit; PreGM, pre-granulocyte/macrophage; Pre-MegE, pre-megakaryocyte/erythrocyte; MkP, megakaryocyte progenitor. g, Representative staining and quantification of CD34, CD41, CD62P and vWF levels on young and old HSCs. Results are from 2 independent cohorts, with data represented as box and whiskers (min to max) and expressed as fold changes in mean fluorescence intensity (MFI) relative to young HSCs. h, Characteristic expression patterns of lineage determinant genes in the droplet-based scRNAseq young and old LK/LSK dataset shown in Fig. 4b. Cells in the UMAP were colored according to the expression levels of the indicated genes. Color scheme is based on ln scale of normalized counts from the indicated minimum (gray) to maximum (red) value in the scale. i, IPA Upstream Regulators analysis of young and old populations from the droplet-based scRNAseq young and old LK/LSK dataset shown in Fig. 4b filtered on cytokines and growth factors. Data are means ± S.D. except when indicated; *p = 0.05, **p = 0.01, ***p = 0.001.

Fig 5: Inflammatory nature of the old BM niche.a, Differentially secreted cytokines in young and old BM fluid measured by 200-plex array and clustered based on broad biological functions (n = 5). b, Changes in pro-inflammatory cytokines in young and old BM fluids. Results are from 3 independent cohorts. c, qRT-PCR-based analyses of Il1a, Il1b and Tnf expression in old BM cells and stromal fractions (n = 6). Results are represented as violin plots and are expressed as fold change compared to their respective young counterpart. Neu, Mac-1+/Ly-6G+/Ly-6Cmid neutrophil; Mac, Mac-1+/Ly-6G-/Ly-6C- macrophage; B cell, B220+/CD19+ B cell; T cell, CD4+/TCRß+ T cell; Marrow, Ter119-/CD45- central marrow fraction; Endo, Ter119-/CD45- endosteal fraction. d, Violin plots representation of Il1a, Il1b, and Il1rn expression in the indicated young and old mesenchymal and endothelial like groups. Results are from the plate-based scRNAseq dataset; *padj = 0.05, ** padj = 0.01, *** padj = 0.001. e, Caspase 1 activity (aCASP1) in the indicated young and old BM and stromal populations with representative FACS plots (top) and quantification (bottom). f, Experimental scheme for the indicated co-culture experiments showing the effects of young and old stroma on young HSC expansion as dependent on IL-1ß. Data are means ± S.D.; *p = 0.05, **p = 0.01, ***p = 0.001.