Fig. 3

FSCN1 promotes chondrocyte shift towards dedifferentiation phenotype via inducing actin polymerization and activating ALK1/Smad1/5 signaling. a Representative images of phalloidin staining of F-actin structures (green) and IF staining for globular (G) actin (red) in chondrocytes with control, IL-1β (10 ng/mL), IL-1β plus FSCN1 knockdown or IL-1β plus FSCN1 overexpression treatment for 24 h. b G-/F-actin ratio quantified by western blotting analysis in all groups (n = 3). c Representative images, alcian blue staining and absorbance quantification of high-density pellet cultured chondrocytes with control, IL-1β (10 ng/mL), IL-1β plus FSCN1 knockdown or IL-1β plus FSCN1 overexpression treatment for 10 days. d Cell extracts were subjected to immunoprecipitation with an anti-DCN antibody. The immunoprecipitate–protein complex was separated with SDS–PAGE and the gel was then stained with silver stain. The identified peptide sequences of FSCN1 by LC–MS/MS analysis are shown. e FSCN1 or DCN was immunoprecipitated from chondrocytes with an anti-FSCN1 or anti-DCN antibody. The presence of FSCN1 and DCN in these immunoprecipitates was evaluated by immunoblotting. f Co-localization of FSCN1 (green) and DCN (red) in chondrocytes with control (saline) or IL-1β (10 ng/mL) treatment for 24 h was analyzed by confocal microscopy. g Western blot analysis of FSCN1, DCN, Collagen type II and III, p-Smad1/5 and Smad1/5 in chondrocytes treated with control, IL-1β (10 ng/mL), IL-1β plus FSCN1 knockdown and with IL-1β plus FSCN1 overexpression for 24 h (n = 3). h Chondrocytes with control, FSCN1 knockdown, or FSCN1 overexpression were subjected to immunoprecipitation with an anti-DCN antibody. The presence of TGF-β1 and DCN in immunoprecipitates was evaluated by immunoblotting. Western blot analysis of FSCN1, DCN, Collagen type II and III, p-Smad1/5 and Smad1/5 (i), IF staining and quantification of Collagen type II (green) and III (red) (j), alcian blue staining and absorbance quantification (k) in chondrocytes with control, IL-1β (10 ng/mL), IL-1β plus FSCN1 knockdown or IL-1β plus FSCN1 and DCN knockdown, IL-1β plus FSCN1 overexpression, IL-1β plus FSCN1 overexpression and LDN-193719 (5 μg/mL) treatment for 48 h (n = 3). Scale bars, 50 μm. All data are presented as means  ± SEM