Figure 4. Oleuropein modulates the Cx43 promoter activity in chondrocytes. (A) Treatment with 10 μM oleuropein for 2 h decreases Cx43 protein levels in T/C-28a2 cells (n=4 independent experiments, Student’s t test, P=0.0012), but this effect was not observed in the same cell line overexpressing Cx43 (pIRES-Cx43)(n=3 independent experiments, Student’s t test, P=0.0624). (B) Luciferase reporter assay indicating that oleuropein inhibits Cx43 promoter activity. The graphs indicate the normalized luminescence activity in the T/C-28a2 transfected with a pGL3-basic plasmid containing 300 base pairs of Cx43 promoter ligated to the luciferase gene. Cells were cultured in DMEM with 10% FBS (UT) and with 5 μg/ml oligomycin or 10 μM oleuropein for 1 h as indicated (n=4 independent experiments; one-way ANOVA, P=0.0012). On the right, Cx43 gene expression under 5 μg/ml oligomycin and 10 μM oleuropein treatment in OACs treated for 1 h (n=4 independent experiments; one-way ANOVA, P=0.0002). Data were normalized to HPRT-1 levels. (C) Immunofluorescence assays of Cx43 in OACs treated with 10 μM oleuropein or 5 μg/ml oligomycin for 1 h. Data were normalized to the untreated condition (n=3 independent experiments; one-way ANOVA, P<0.0001). Data is expressed as mean±SD; *P<0.05, **P<0.01 and ***P<0.0001.