Fig 2: Evaluation and analysis of OVX-induced model via micro CT mainly(A) Weight change diagram of C57BL/6J mice before and after OVX surgery.(B and C) Tissue total RNA was extracted from Mice forelimb, and the mRNA expression levels of AhR, CTSK, NFATc1, DC-STAMP, ACP5, c-Fos, and ATP6v0d2 were displayed by qPCR, normalized to GAPDH.(D and E) Mice forelimb bone tissue was isolated of from each experimental group. Adding tissue lysate to extract tissue protein, AhR specific primary antibody was used to show the results by western blot.(F and G) The micro-CT was used to scan tibias of each mouse, and trabecular bone volume per total volume (BV/TV) was recorded for each specimen. And the relative data are compared. The Data were presented as means ± SEM; n = 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Fig 3: 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 4: SR1 inhibits RANKL-induced osteoclastogenesis in a concentration-dependent manner(A) The chemical structural formula of StemRegenin 1.(B) The cell viability of different concentration gradients SR1-stimulated BMMs after 48 h and 96 h.(C) The BMMs were induced by 25 ng/mL MCSF and 50 ng/mL RANKL for indicated periods, the mRNA expression levels of AhR was detected by qPCR.(D) The BMMs were induced by 25 ng/mL MCSF and 50 ng/mL RANKL and stimulated with SR1 at indicated concentrations for 4 days, the mRNA expression levels of AhR was detected by qPCR.(E) The BMMs were induced by 25 ng/mL MCSF and 50 ng/mL RANKL and stimulated with SR1 at indicated concentrations for 4 days, the mRNA expression levels of osteoclasts-specific genes, including NFATc1, ACP5, DC-STAMP, ATP6v0d2, CTSK, were detected by qPCR, and normalized relative to GAPDH.(F) The BMMs were induced by 25 ng/mL MCSF and 50 ng/mL RANKL and stimulated with SR1 at indicated concentrations for 4 days. Then the western blot was used to show protein expression levels of NFATc1, CTSK, ACP5, and AhR. And quantitative analysis of protein expression levels, normalized to β-actin.(G) BMMs were induced by 25 ng/mL MCSF and 50 ng/mL RANKL, and stimulated with SR1 at indicated concentrations (3, 6, 8, 10 μM) for 7 days. Osteoclasts formation results were shown using TRAP staining, scale bar, 100 μm.(H) Quantitative results of multi-nuclear TRAP-positive cell numbers and the area occupied. We divided the multi-nuclear TRAP-positive cells into three groups (3–5, 5–10, and 10-) according to the number of nuclei, and the TRAP-positive cells number of each group was analyzed.(I) The BMMs were stimulated with 10 μM SR1. The detection of the NFATc1 and CTSK expression level carried out immunofluorescence, 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 5: 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)