Fig. 5

FSCN1 inhibitor NP-G2-044 protects chondrocytes against dedifferentiation in vitro via suppressing ALK1/Smad1/5 signaling. a IF staining of FSCN1 (red) and phalloidin (green) staining for F-actin structures in chondrocytes treated with control, IL-1β (10 ng/mL), or IL-1β plus NP-G2-044 (10 μmol/L) for 24 h. Heat map (b) and volcano plot (c) showing differentially-expressed genes (DEGs) (fold change > 2 or <0.5) from RNA-sequencing of chondrocytes treated with IL-1β or IL-1β plus NP-G2-044; n = 3 per condition and seven independent experiments were analyzed. KEGG pathway (d) and GSEA (e) analysis for DRGs demonstrating TGF-β and Wnt signaling pathway enrichment in chondrocytes treated with IL-1β or IL-1β plus NP-G2-044. f Images of optical microscopy, phalloidin staining for F-actin structures (green) and IF staining for globular actin (red) in chondrocytes treated with control, IL-1β (10 ng/mL), or IL-1β plus NP-G2-044 (10 μmol/L) for 24 h. g G-/F-actin ratio quantified by western blotting analysis; n = 3 independent experiments. Alcian blue staining and absorbance quantification (h), IF staining and quantification of Collagen II (green) and III (red) (i), IF staining and quantification of p-Smad1/5 (green) and β-catenin (red) (j) in chondrocytes treated with control, IL-1β, or IL-1β plus NP for 24 h (n = 3). Relative mRNA expression of chondrogenic and dedifferentiation genes (k), Western blot analysis of FSCN1, DCN, Collagen type II and III, p-Smad1/5 and Smad1/5 (l) in chondrocytes treated with control, IL-1β, or IL-1β plus NP-G2-044 for 24 h (n = 3). Scale bars, 50 μm. All data are presented as means ± SEM