Fig 2: Genetic and pharmacological validation that FPR1 mediates 25-HC signalling.a Representative fluorescence and bright-field images of ADSCs transduced with LV-NC, LV-FPR1, sh-NC, or sh-FPR1. b Immunoblot of FPR1 with Tubulin as loading control and corresponding densitometric quantification in the groups shown in (a) (mean ± SEM; n = 3). c qRT–PCR measurement of FPR1 mRNA levels in LV-NC, LV-FPR1, sh-NC and sh-FPR1 cells, normalised to GAPDH (mean ± SEM; n = 3). d Immunoblot analysis of PI3K/AKT/GSK3β pathway proteins (PI3K, p-AKT, AKT, p-GSK3β, GSK3β; GAPDH as loading control) in the indicated 25-HC stimulation, FPR1 knockdown and lentiviral rescue groups (labels above lanes); the same colour-coding is used to denote corresponding groups across panels (d–g) (mean ± SEM; n = 3). e RT–qPCR analysis of SOX9, ACAN and COL2A1 mRNA expression in the indicated groups (mean ± SEM; n = 3). f Immunoblot detection of chondrogenic markers (SOX9, ACAN, COL2A1; GAPDH loading control) and corresponding densitometric quantification for the conditions indicated (mean ± SEM; n = 3). g Alcian Blue staining, Safranin O staining and COL2A1 immunofluorescence (with DAPI and merged images) for the indicated groups. h Immunoblot analysis and densitometric quantification of PI3K/AKT/GSK3β pathway proteins in the NC, cyclosporin H plus HCH6-1 (cyH + HCH), fMLF and fMLF + cyH + HCH groups; the same colour-coding is used to denote corresponding groups across panels (h–k) (mean ± SEM; n = 3). i qRT–PCR validation of SOX9, ACAN and COL2A1 mRNA expression in the indicated groups (mean ± SEM; n = 3). j Immunoblot detection of SOX9, ACAN and COL2A1 (GAPDH loading control) and densitometric quantification in the indicated groups (mean ± SEM; n = 3). k Alcian Blue staining, Safranin O staining and COL2A1 immunofluorescence (with DAPI and merged images) in the indicated groups.

Fig 3: Pharmacological validation of the 25-HC–FPR1–AKT axis in ADSCs.a Western blot of total PI3K, phospho-AKT (Ser473), total AKT, phospho-GSK3β (Ser9) and total GSK3β in sh-NC and sh-CH25H cells treated for 7 d with DMSO, 4 µM 25-HC, or 4 µM 25-HC + 10 µM cyH + HCH (cyclosporin H + HCH6-1); bands quantified in ImageJ and normalised to GAPDH (mean ± SEM; n = 3). b RT-qPCR of SOX9, ACAN and COL2A1 mRNA on day 21 in sh-NC and sh-CH25H cells exposed to DMSO, 4 µM 25-HC, or 4 µM 25-HC + 5 µM MK + LY (MK2206 + LY294002); values normalised to GAPDH (mean ± SEM, n = 3). c Western blot of SOX9, ACAN and COL2A1 proteins under the conditions in (b); bands quantified in ImageJ and normalised to GAPDH (mean ± SEM; n = 3). d Alcian Blue and Safranin O staining of extracellular matrix after 21 d for the treatments in (b); scale bar, 200 µm. e COL2A1 immunofluorescence (green) with DAPI nuclear counter-stain (blue) on day 21 for the treatments in (b); scale bar, 50 µm.

Fig 4: SPR validation of 25-HC binding to FPR1 and MD analyses of complex stability, binding energetics, key interactions and TM6 dynamics.a Schematic illustration of the SPR assay showing 25-HC captured by an FPR1-coated sensor chip. Panel a was created in BioRender. YANG, W. (2026) https://BioRender.com/0pwuazm. b SPR sensorgrams of 25-HC (0.0977–12.5 μM) flowing over immobilised His-tagged FPR1, with response units (RU) plotted versus time. c Steady-state affinity plot (equilibrium RU versus ligand concentration) fitted with a 1:1 Langmuir model. d All-atom MD simulation system of the 25-HC–FPR1 complex embedded in a lipid bilayer and solvated, shown in side and top views. e Backbone RMSD traces of apo-FPR1, 25-HC, and the 25-HC–FPR1 complex over a 100-ns trajectory. f Radius of gyration (Rg) of the complex versus simulation time. g Solvent-accessible surface area (SASA) of the complex versus simulation time. h Per-residue RMSF of FPR1 Cα atoms over the 100-ns trajectory, with major loop regions (ECLs/ICLs) annotated across the 7TM domain. i MM/GBSA binding-free-energy components and total binding-free energy (ΔG_total) for the complex. j Per-residue MM/GBSA decomposition highlighting key pocket residues contributing to binding. k Two-dimensional free-energy landscape (FEL) projected onto the first two principal components (PC1/PC2). l Three-dimensional representation of the same FEL (Gibbs free energy) derived from PC1/PC2. m Representative minimum-energy snapshot showing 25-HC bound within the FPR1 pocket. n Magnified view of the ligand-binding pocket highlighting residues lining the binding site. o Hydrogen-bond interaction schematic between 25-HC and FPR1 (dashed lines indicate hydrogen bonds). p Hydrogen-bond occupancy summary for major hydrogen-bond pairs during the 100-ns simulation. q Time-series of hydrogen-bond angles for the highlighted hydrogen-bond pairs over 100 ns. r Time-series of donor–acceptor distances for the same hydrogen-bond pairs over 100 ns. s TM6 outward-displacement distance over time, defined as the Cα–Cα separation between R3.50 on TM3 (Arg165) and L6.34 on TM6 (Leu258). t TM6 tilt (outward-swing) angle trajectory, defined as the angle between the TM6 helical-axis vector and the membrane normal (z-axis).

Fig 5: AKT activation rescues 25-HC signalling blocked at the FPR1 level in wild-type ADSCs.a RT-qPCR analysis of SOX9, ACAN and COL2A1 mRNA on day 21 in wild-type ADSCs treated with DMSO, 4 µM 25-HC, 4 µM 25-HC + 10 µM cyH + HCH (cyclosporin H + HCH6-1) or 4 µM 25-HC + 10 µM cyH + HCH + 10 µM SC + 740 (SC-79 + 740 Y-P); values normalised to GAPDH (mean ± SEM, n = 3). b Western-blot detection of total PI3K, phospho-AKT (Ser473), total AKT, phospho-GSK3β (Ser9) and total GSK3β on day 7 under the same treatments as in (a); bands quantified in ImageJ and normalised to GAPDH (mean ± SEM; n = 3); (c) Alcian Blue and Safranin O staining of extracellular matrix after 21 days for the treatments in (a); scale bar, 200 µm. d COL2A1 immunofluorescence (green) with DAPI nuclear counter-stain (blue) on day 21 for the treatments in (a); scale bar, 50 µm.