Figure 3. Oleuropein treatment enhances chondrocyte redifferentiation. (A) Immunohistochemistry of Col2A1 (4-6 independent experiments; one-way ANOVA, P=0.0019) and toluidine blue staining of proteoglycan subunits (n=6 independent experiments; one-way ANOVA, P=0.059) indicate significant enrichment in ECM components in OACs micromasses grown in 3D culture for 30 days in chondrogenic medium (CM) when supplemented with 10 μM oleuropein (Oleu) or OE. (B) Cx43 protein levels detected by western blot (and normalized to Ponceau staining) are reduced when OACs micromasses are exposed to CM supplemented with 10 μM oleuropein or OE for 21 days (n=3 independent experiments; one-way ANOVA, P=0.0328). (C) Oil red staining showing reduced OACs dedifferentiation upon exposure to Oleu or OE in adipogenic medium (n=5 independent experiments; one-way ANOVA, P=0.0001). (D) Nuclear levels of Twist-1 were decreased in OACs cultured with 10 μM oleuropein for 2 h. Lamin A was used as a loading control (n=3 independent experiments; Student’s t test, P=0.001). (E) Cx43 protein levels in primary OACs after 1-h treatment with oleuropein or oligomycin. Western blot represents n=4 independent experiments. Quantification is shown on the right (one-way ANOVA, P=0.0036). On the right, immunofluorescence for Twist-1 (red) in primary OACs treated with 5 μg/ml oligomycin and 10 μM oleuropein for 1 h. The graph represents the percentage of cells with Twist-1 nuclear localization (n=4 independent experiments; one-way ANOVA, P=0.0067). (F) The mRNA expression of the EMT markers Twist-1, N-Cadherin and Vimentin in OACs treated with 10 μM oleuropein for 2 h. Data were normalized to HPRT-1 levels. n= 5 independent experiments; Student’s t test: P<0.0001 (Twist-1), P= 0.0011 (N-Cad), P=0.0209 (Vim). Data is expressed as mean±SD; *P<0.05, **P<0.01 and ***P<0.0001.