Fig 2: AB-EVs promote bone-fat imbalance in young and aged mice.a Schematic diagram of the experimental design for assessing the effects of YB-EVs and AB-EVs on bone phenotypes in young and aged mice. b µCT-reconstructed images of femurs. Scale bars: 1 mm. c Quantification of Tb. BV/TV, Tb. N, Tb. Th, and Tb. Sp. n = 10 biologically independent animals per group. d Ultimate load values of femurs. n = 10 biologically independent animals for young mice. n = 6 biologically independent animals for aged mice. e–f, Calcein (green) double labeling of trabecular bones (e) and quantification of BFR/BS and MAR (f). Scale bar: 25 µm. n = 5 biologically independent animals per group. g–h, PLIN immunofluorescence staining images of femur sections (g) and quantification of the number of PLIN+ (red) adipocytes in bone marrow (h). Scale bar: 100 µm. n = 6 biologically independent animals per group. i qRT-PCR for Ppar? expression in femurs. n = 9 biologically independent animals per group. j–k, OCN immunohistochemical staining images (j) and the number of OCN-stained (brown) osteoblasts (N. OBs) on the trabecular bone surface (BS) (k). Scale bar: 50 µm. n = 6 biologically independent animals per group. l ELISA for serum OCN. n = 10 biologically independent animals per group. All experiments were performed with at least five biological replicates per group without independent repetition. Data were presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Bonferroni post hoc test. Source data are provided as a Source Data file.

Fig 3: Muscle PGC-1a overexpression prevents the accumulation of intramuscular adipocytes(A) Representative images of H&E staining (scale bar: 200 µm).(B) Heatmap showing relative expression of Myh3, Plin1, Adipoq, Fapb4, Cd55 and Cd142 genes by RT-qPCR quantification of 3-month-old WT and MCK-PGC-1a mice injected with 50% glycerol (Gly) at 21 dpi. Data are normalized by Actb expression and displayed as relative expression (2–??Ct; mean) vs WT-Sham group. Significance of the differences: *p<0.05 vs WT-Sham or MCK-PGC-1a-Sham group (two-way ANOVA+ Holm-Šídák test; n = 7 mice/group).(C) Representative images of ORO staining (scale bar: 200 µm).(D and F) Representative images of perilipin/Hoechst immunostaining of TA muscle (scale bar: 500 µm) of (D) 3-month-old WT and MCK-PGC-1a mice injected with Gly at 21 dpi and (F) 12-month-old WT and MCK-PGC-1a mice injected with BaCl2 at 14 dpi. White dotted lines indicate the boarders of the TA muscle. In D, red-doted rectangles highlight the same area shown in panels A and C.(E and G) Densitometric quantification of Alexa488 signal, proportional to anti-perilipin antibody labeling. Significance of the differences: *p<0.05 (Student’s t test; n = 7 mice/group).

Fig 4: Increased fatty acid oxidation in Nf1Myf5 muscle. (A) Depiction of significantly deregulated proteins related to cellular lipid uptake and breakdown. Proteome analysis of p21 muscle, genotypes are indicated. (B) Gene set enrichment analysis (GSEA) of control vs. Nf1Myf5 muscle transcriptome data for the terms ‘fatty acid beta-oxidation’ and ‘fatty acid catabolic process’. (C) Heat map of differentially expressed genes related to fatty acid metabolism (control vs. Nf1Myf5 muscle). (D) RT-qPCR analysis in control vs. Nf1Myf5 muscle of selected genes related to fatty acid uptake, transport, and catabolism (n = 3 animals per genotype). (E) Perilipin (PLIN, red) and MyHC (green) immunolabelling on 12-week-old control vs. Nf1Myf5 muscle. Overview tile scan images left, magnifications showing subcutaneous adipose tissue and adjacent muscle tissue shown right. (F) RT-qPCR analysis of Pparg in control vs. Nf1Myf5 muscle (n = 3 animals per genotype). (G) Western blot analysis for PPAR? on control vs. Nf1Myf5 muscle. (H) GSEA of control vs. Nf1Myf5 muscle transcriptome data for the term ‘adipogenic targets of PPARG’. (I) RT-qPCR analysis for Adipoq, Il6, and Fgf21 on control vs. Nf1Myf5 muscle (n = 3 animals per genotype). Error bars represent standard error of means. P-value was calculated by two-sided unpaired t-test.

Fig 5: ALE down-regulates AB-EVs release and attenuates bone-fat imbalance and VD3-induced vascular calcification in aged OVX mice.a Total protein contents of EVs isolated from the conditioned media of osteoclasts treated with solvent (OC-CM), ALE (OCALE-CM), AB (OCAB-CM), or AB + ALE (OCAB+ALE-CM). n = 5 biologically independent samples per group. b–d, Quantification of the percentages of ARS+ (b) and ORO+ (c) areas in BMSCs with different treatments under osteogenic or adipogenic induction, or ARS+ areas (d) in VSMCs with different treatments under osteogenic induction. n = 5 biologically independent cells per group. e–f µCT-reconstructed images of femurs from 16-month-old Sham or OVX mice in different groups (e) and quantification of Tb. BV/TV, Tb. N, Tb. Th, and Tb. Sp (f). Scale bars: 1 mm. n = 5 biologically independent animals per group. g–h PLIN immunofluorescence staining images of femur sections (g) and quantification of the number of PLIN+ (red) adipocytes in bone marrow (h). Scale bar: 100 µm. n = 5 biologically independent animals per group. i qRT-PCR for Ppar? expression in femurs. n = 5 biologically independent animals per group. j–l OCN immunostaining images (j), quantification of the number of OCN-stained (brown) osteoblasts on BS (k), and ELISA for OCN (l). Scale bar: 50 µm. n = 5 biologically independent animals per group. m–n ARS staining images (m) and quantification of the percentage of ARS+ areas (red; n). Scale bar: 200 µm. n = 5 biologically independent animals per group. o Vascular calcium content measurement. n = 5 biologically independent animals per group. p–q, RUNX2 immunostaining images (p) and quantification of the percentage of RUNX2+ (red) areas (q). Scale bar: 200 µm. n = 5 biologically independent animals per group. r qRT-PCR for Alpl expression. n = 5 biologically independent animals per group. Experiments in b–d were repeated independently three times with similar results. The illustrated results represented one of the three independent experiments. The other experiments were performed with at least five biological replicates per group without independent repetition. Data were presented as mean ± SD. Statistical significance was determined by two-way ANOVA with Bonferroni post hoc test. Source data are provided as a Source Data file.