Fig 2: Galectin-8 drives the uptake of CD166 and induces the recruitment of Endophilin-A3 to endocytic sites at the plasma membrane.HeLa cells. a Co-incubation with 5 µg ml-1 anti-CD166 antibody and 50 nM fluorescent Galectin-8 (Gal8) for 10 min at 37 °C. Graph, quantifications of co-localization (n = 15 images with 5–15 cells per image). Three independent experiments. ****P < 0.0001 (two-tailed unpaired t test with equal variances). Galectin-1 (Gal1) condition, Supplementary Fig. 9c. b Cells treated with control (siCtrl, red) or Gal8 (siGal8, blue) siRNAs incubated with 5 µg ml-1 anti-CD166 antibody for 10 min at 37 °C in various conditions (graph: ±10% FBS, ±purified Gal8). Intracellular accumulation of anti-CD166 quantified from confocal images. n cells, four independent experiments. NS not significant. ****P < 0.0001 (Kruskal–Wallis test with Dunn’s multiple comparison post hoc test, two-sided). c Cells stably expressing endoA3-GFP incubated with 50 nM fluorescent Gal8 for 10 min at 37 °C. White arrows, co-localization. Graph, number of co-localization events per cell. Number of cells: Gal8, n = 35; Gal1, n = 31 cells. Three independent experiments. ****P < 0.0001 (two-tailed unpaired t test with Welch’s correction). Gal1 condition, Supplementary Fig. 9f. d–f Gal8-induced recruitment of endoA3-GFP at plasma membrane. d Fluid-FM coupled to the confocal setup. Fluorescent galectin-coated gold nanoparticles trapped by microchanneled cantilever and approached to endoA3-GFP-expressing cells. Simultaneous monitoring of galectin (red) and endoA3 (green) fluorescence by fast-scanning confocal microscopy. e Representative images of Fluid-FM/confocal experiments (ten independent measurements). t = 0 s, initial cell exposure to Gal8. TL, transmitted light (cantilever). Insets: area surrounding the beads at the indicated time points. Graph: green fluorescence intensity around beads versus time. Images and plots for Gal1, dye control, bare probe, and endoA2-GFP controls, respectively, Supplementary Fig. 9i–l. f Quantification of fluorescence over time (slopes of linear regressions fitted on intensity vs. time curves). n-independent measures. NS not significant. **P < 0.01, ***P < 0.001 (two-tailed unpaired t tests). Scale bars, 10 µm (a), 1 µm (c), and 5 µm (e). Data are mean ± s.e.m. (a, c, f) or median ± 95% CI. b Source data are provided as a Source Data file.

Fig 3: iCSFCs have fibroblast properties. (A) Gene expression of fibroblast cell markers was examined by RT-PCR. Most fibroblast genes were expressed in iCSFCs, with the exception of Acan, MMP13 and Sox6. GAPDH was used as an internal control. (B,C) Immunocytochemistry analysis of iCSFCs for fibroblast marker proteins. Immunostaining was used to indicate fibroblast markers (red) in CFF-3 cells (B) and iCSFCs (C), including ALCAM, Col1A1, HSP47 and S100A4. Cell nuclei were labeled with DAPI (Scale bars, 50 µm).

Fig 4: (A) Flow cytometric characterization of IFPSCs at day 14 (P2), with unstained cells serving as the negative control. The cells were characterized according to the surface antigen expression of CD90, CD105, CD166 and CD45, as well as the transcription factor Sox2 and early neuronal marker nestin. (B) Immunocytochemical analysis of IFPSCs for stem cell markers at day 14 (P2; magnification, ×20). FITC-stained images show the positive expression of the following stem cell-specific markers: (a) Nucleostemin, (b) Nanog, (c) CD166, (d) CD105 and (e) CD13, with (f) unstained cells serving as the negative control. Scale bar, 50 µm. IFPSCs, infrapatellar fat pad-derived stem cells; FITC, fluorescein isothiocyanate; PE, phycoerythrin; APC, allophycocyanin; Sox2, SRY-box 2.

Fig 5: Downregulation of Cx43 by oleuropein decreases GJIC and improves the phenotype of OACs. (A) Oleuropein (Oleu) treatment significantly decreases GJIC evaluated by an SL/DT assay when OACs were exposed with this molecule for 2 h (top, n=6 independent experiments; Student’s t test, P<0.0001). The results were confirmed by calcein transfer by flow cytometry (n=4 independent experiments; Student’s t test, P=0.0037). (B) Graph showing the effect of oleuropein on GJIC when healthy chondrocytes (N) were exposed to 10 µM oleuropein compared with OACs (n=5 independent experiments; one-way ANOVA, P=0.0004). (C) OACs cultured for 7 days with 10 µM Oleu showed reduced expression of the mesenchymal markers CD105 and CD166, analyzed by flow cytometry. Student’s t test, P=0.0039 (CD105) and P=0.0022 (CD166), n=6 independent experiments. CD166 levels were also analyzed by western blot (n=3 independent experiments, Student’s t test, P=0.0046). (D) Downregulation of Cx43 increased Col2A1, detected by immunofluorescence in OACs treated with 10 µM oleuropein. Graphs represent the corrected total cell fluorescence (CTCF) of Cx43 and Col2a1 (n=4 independent experiments). Student’s t test, P<0.0001 (Cx43), and P=0.0007 (Col2a1). (E) mRNA levels of IL-1ß, IL-6, COX-2 and MMP-3 of OACs cultured in normal medium (UT) exposed to 10 µM oleuropein for 2 h. n=4–7 independent experiments. Student’s t test: P= 0.033 (IL-1ß), P<0.0001 (IL-6), P=0.1013 (COX-2), P=0.0466 (MMP-3). (F) IL-6 detected by ELISA when OACs were treated with oleuropein for 72 h (n=4 independent experiments, Student’s t test, P=0.0345). IL-6 (n=3 independent experiments) and COX-2 (n=4 independent experiments) protein levels detected by western-blot in OACs treated with 10 µM oleuropein for 72 h. Student’s t test, P=0.0193 (IL-6), P=0.0141 (COX-2). Data is expressed as mean±SD; *P<0.05, **P<0.01 and ***P<0.0001.