Fig 2: SR 1 inhibits RANKL-induced osteoclastogenesis in a time-dependent manner(A) For 0, 1, 3, 5 days with or without 10 μM of SR1, the treatment of BMMs was carried out with RANKL, and the osteoclastogenesis related genes expression level was detected by qPCR.(B and C) TFor 0, 1, 3, 5 days with or without 10 μM of SR1, the treatment of BMMs was carried out with RANKL, and Western blot was used to show protein expression levels of NFATc1, CTSK, ACP5, c-Fos, AHRR, and AhR. And quantitative analysis of protein expression levels, normalized to GAPDH.(D) Patterns of BMMs stimulated with SR1 at different times during the 7 days of BMMs differentiation.(E and F) BMMs were induced by 25 ng/mL MCSF and 50 ng/mL RANKL, and stimulated with 10 μM SR1 at different stages (1–3, 3–5, 5–7 Day). Quantitative results of multi-nuclear TRAP-positive cell numbers and the area occupied, Scale bar, 100 μm. Data were presented as means ± SEM; n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig 3: PCLAF binds to the ADGRL2 receptor of BMAds to accelerate skeletal aging. a LC-MS/MS analysis of identified ADGRL2. b Co-immunoprecipitation (IP) analysis of binding between His-PCLAF and Flag-ADGRL2. c, d Representative images (c) and quantification of SA β-gal (d) staining of BMSCs-derived adipocytes transfected with siRNA-Adgrl2 or siRNA-NC in the presence or absence of rPCLAF (n = 3). e Representative western blot of AKT, mTOR protein/phosphorylation level of BMSCs-derived adipocytes transfected with siRNA-Adgrl2 or siRNA-NC in the presence or absence of rPCLAF. f Pattern diagram of experimental design for transplantation of BMAds from AAV-Fabp4-Scramble or AAV-Fabp4-ShAdgrl2 mice and then treated with rPCLAF or PBS. g Representative images of Micro-CT of mice in f (n = 5). h–k Quantitative analysis of Tb.BV/TV (h), Tb.Th (i), Tb.N (j), Tb,Sp (k) (n = 5). l, m Representative images of co-localization staining of OCN (l) and Ctsk (m) with senescent markers (p21 and γH2AX) (n = 5). n, o Quantification of double positive cells per mm2 tissue area, shown as N. Ocn+γH2AX+ cells (n) and N. Ctsk+p21+ cells (o) (scale bar, 50 μm; n = 5). Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA (d, h–k, n, o)

Fig 4: Gene profiling analysis of PRDX1 during osteoclastogenesis(A) The period for osteoclast differentiation was divided into four distinct time periods (T1 to T4). PRDX1 (200 nM) was treated during each period, and TRAP-positive multinucleated cells were counted.(B) Heatmap shows mRNA expression profiles of PRDX1 during osteoclastogenesis. K-means (K = 5) clustering of 4,328 differentially expressed genes (DEGs) in any pairwise comparison among four conditions (left panel). The clusters are indicated on the left. The right panel shows a heatmap showing the P-value significance and gene numbers of gene ontology (GO) analysis for genes in each cluster.(C) GSEA plot shows the increased gene expression of cell surface toll-like receptor signaling pathway in PRDX1 (PRDX1 + no RANKL condition) compared to control (no PRDX1 + no RANKL). Cfb and Ccl5 expression levels were measured by RT-qPCR using specific primers.(D) GSEA plot shows the decreased gene expression of the regulation of osteoclast differentiation in PRDX1 + RANKL treatment compared to RANKL treatment. mRNA expression levels of Ctsk, Dcstamp, and Nfatc1 were measured by RT-qPCR. The data are presented as the mean ± SD values from three independent experiments; P-value was examined by one-way ANOVA with Tukey’s multiple comparisons test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Fig 5: PCLAF-mediated BMAds senescence induces bone loss. a Representative image of Micro-CT of mice injected with rPCLAF or PBS (n = 6). b–e Quantitative analysis of Tb.BV/TV (b), Tb.Th (c), Tb.N (d), Ct.Th (e) (n = 6). f–h Representative images of osteocalcin (OCN) staining (f) and quantification (g, h) of the number of OCN+ cells (red arrows) in femurs (scale bar, 50 μm; n = 6). i Pattern diagram of experimental design for BMAds transplantation. j Representative images of Micro-CT of mice transplanted with PBS-treated or rPCLAF treated BMAds (n = 5). k–n Quantitative analysis of BV/TV (k), Tb.Th (l), Tb.N (m), Tb.Sp (n) (n = 5). o–q Representative images of co-localization staining of OCN (o), Ctsk (p) and leptin (q) with senescent markers (p21, γH2AX) (scale bar, 50 μm; n = 5). r–t Quantification of numbers of double-positive cells per mm2 tissue area, shown as N. OCN+ γH2AX+ cells (r), N. Ctsk+ p21+ cells (s) and N. Leptin+ γH2AX+ cells (t), respectively (n = 5). u, v Representative images of calcein double-labeling (u) and analysis of mineral apposition rates (MARs) (v) of trabecular bone in femurs (scale bars, 50 mm, n = 5). Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t-test (b–e, g, h, k–n, r–t, v)