Figure 6.

Dex induces calcification of ECM partially by activating AKT signaling. (A) Alizarin red staining to assess the extent of extracellular matrix calcification of Dex at different concentration in primary chondrocytes. (B) Alizarin red absorbance was detected by microplate reader at 562 nm (n = 3). (C) Alizarin red staining to assess the extent of extracellular matrix calcification of 10 nM Dex with or without AKT inhibitor LY294002 in primary chondrocytes. (D) Alizarin red absorbance was detected by microplate reader at 562 nm (n = 6). (E) Cell lysates of primary chondrocytes were analyzed by western blotting using antibodies of AKT, P-AKT³⁰⁸, P-AKT⁴⁷³ (n = 3). (F) IHC analysis of P-AKT³⁰⁸ protein expression in articular cartilage of mice at 4 weeks after DMM surgery with or without Dex, expansion of the region occupied by articular cartilage, Scale bar: 100 µm. (G) The percentage of cells that are positive for P-AKT³⁰⁸ in the articular cartilage were calculated (n = 4). Differences between two groups were evaluated using Student's unpaired t-test, and comparisons of multiple groups were evaluated using analysis of variance (ANOVA) followed by Tukey's test. Data were expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.