Fig 2: Increased endogenous or exogenous FABP4 protein both exacerbates OCs differentiation.a TRAP staining of BMMs after the treatment of rmFABP4 (25 ng/mL or 50 ng/mL) or simultaneously with FABP4 inhibitor BMS (10 μM or 30 μM). Scale bar, 500 μm. n = 4 for each 96-well plate. b Quantitative cell number of OCs in (a). n = 4 for each 96-well plate. c–d Western Blotting strips (c) and its quantitative analysis results (d) indicated treatment with FFA mixture will upregulate the intracellular FABP4 in BMMs. n = 3 independent experimental samples per group. e TRAP staining of BMMs after the treatment of FFA mixture (200X or 400X dilution) or simultaneously with FABP4 inhibitor BMS (10 μM or 30 μM). Scale bar, 500 μm. f Quantitative cell number of OCs in (e). n = 6 for each 96-well plate. g SEM results of bovine bone slices show that bone resorption experiments were conducted with OCs and treated with rm-FABP4, FFA, and BMS. Scale bar, 2 μm. Three independent experiments were performed. h TRAP staining of BMMs after treated with different concentration of BMS. Scale bar, 500 μm. n = 6 for each 96-well plate. i Comparison of the IC50 values of ALD and BMS in inhibiting OCs differentiation activity; n = 6 for each 96-well plate. The IC50 values were calculated by log(inhibitor) vs. normalized response - Variable slope. All experiments were run in at least triplicate. All data is presented as mean ± SEM with p-values (b, d, f, i), ns no significance. Data was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test (b, d–f). Source data are provided as Source Data file.

Fig 3: Increased endogenous or exogenous FABP4 protein does not affect the osteogenic differentiation of BMSCs.a Full image of the effect of rmFABP4 (25 ng/mL or 50 ng/mL) or combined with FABP4 inhibitor BMS (5 μM or 10 μM) on BMSCs osteogenic differentiation, assessed by Alizarin Red staining. b Quantitative analysis of (a). n = 6 wells in 6-well-plate per group. c Representative bright-field and GFP images of FABP4 stably overexpressed BMSCs. Scale bar, 200 μm. At least three independent experiments were performed. d Western Blotting indicated the successful overexpression of FABP4 protein in FABP4 overexpressed BMSCs. Three independent experiments were performed. e q-PCR results showed a nearly 3600-fold increase in FABP4 expression in BMSCs. n = 3 independent samples per group. f Full image of the effect of FABP4 overexpressed BMSCs osteogenic differentiation by Alizarin Red staining. g Quantitative analysis of (f). n = 6 wells in 6-well-plate per group. All experiments were run in at least triplicate. All data is presented as mean ± SEM with p-values (b, e, g), ns = no significance. Two-tailed unpaired t test (e) and one-tailed Dunnett’s Multiple Comparison Test (b, g) were performed, and the p-values were adjusted made by multiple comparisons (b, g). Source data are provided as Source Data file.

Fig 4: BMS blocks OCs differentiation via Ca2+-Calcineurin-NFATc1 signaling pathway.a Volcano plot of transcriptome sequencing comparing BMMs vs. OCs and OCs vs. OCs treated with 1 μM BMS. n = 3 independent experimental samples. b Venn diagram showing upregulated genes in BMMs vs. OCs and downregulated genes in OCs vs. OCs + BMS. c KEGG pathway analysis of intersecting genes from the Venn diagram. d Cluster analysis of FPKM values for genes associated with OCs differentiation. e qPCR results of differentially expressed genes mentioned in (d). n = 3 independent experimental samples. f Western Blotting results of OCs differentiation marker proteins. g Quantitative analysis of (f). n = 3 independent experimental samples. h Flow cytometry quantification of intracellular Ca2+ concentration in OCs treated with FABP4 (25 ng/mL), FABP4 (25 ng/mL) + BMS (30 μM or 10 μM) (F + BMS30, F + BMS10), and BMS (1 μM) (BMS1) alone. i In situ fluorescence imaging of intracellular Ca2+ in OCs across different groups. Scale bar, 100 μm. Three independent experiments were performed. j Expression levels of Calcineurin and PLCγ proteins in OCs after BMS treatment. k Quantitative analysis of (j). n = 4 independent experimental samples. l Schematic diagram illustrating the mechanism by which the FABP4 inhibitor suppresses OCs differentiation. Created in BioRender. Li, J. (2025) https://BioRender.com/ha5yaui. All experiments were run in at least triplicate. All data is presented as mean ± SEM with p values (e, g, k), ns = no significance. Data was analyzed by one-tailed Student’s t-test (a, c) and one-way ANOVA followed by Tukey’s multiple comparisons test (e, g, k). Source data are provided as Source Data file.

Fig 5: Oral FABP4 inhibitor BMS increases bone density in OVX mice by inhibiting bone resorption.a Schematic illustration of the animal study groups and experimental procedures. The scissors, ovary and syringe icons are from templates provided by ChemDraw. b–d ELISA detection of serum levels of FABP4 (b) PINP (c) and CTX-1(d) at the endpoint. n = 5 mice per group in (b, d) and 10 mice per group in (c). e Micro-CT scans of the distal femur in each group, with trabecular bone highlighted in blue and the 3D analysis conducted within the defined ROI region. f Coronal micro-CT images of the L4-L5 lumbar vertebrae in each group, with the 3D analysis ROI region highlighted in bright blue. g–i BMD (g) Tb.Th (h) and Tb.N (i) of trabecular bone in the distal femur for each group. j–l BMD (j) Tb.Th (k) and Tb.N (l) in the trabecular bone of the L5 lumbar vertebra. m–n Quantitative TRAP staining results (m) and representative images (n) of demineralized proximal tibia sections in each group. n = 4 mice per group (g–m). Scale bar, 400 μm and 100 μm. All data is presented as mean ± SEM with p values (b–d, g–m). ns no significance. Data was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test (b–d, g–m). Source data are provided as Source Data file.