Ehistatin inhibited dedifferentiation of monolayer-cultured chondrocytes. (a) Primary cultured chondrocytes were cultured in monolayers in the presence or absence of echistatin, and cell morphology was observed 7 days after plating. Scale bar = 100 μm. (b) After macroscopic observation, immunofluorescent staining was performed to evaluate formation of focal adhesion and filamentous actin (F-actin) cytoskeleton. Green fluorescence indicates vinculin localized in focal adhesion, while red indicates phalloidine associated with F-actin. Scale bar = 20 μm. (a), (b) Representative images of five independent experiments. (c) Chondrocytes were cultured in the presence or absence of echistatin for 7 days, and expression of type II procollagen (COL2A1) and aggrecan (ACAN) was evaluated by quantitative PCR, together with that of type I procollagen (COL1A1) and type III procollagen (COL3A1). Results are shown by relative ratios against the cells cultured without echistatin (open bars). Results are mean ± standard error of the mean (SEM) of five independent experiments, each duplicate. **P <0.01 against cells cultured without echistatin. (d) Chondrocytes were cultured for 7 days with or without echistatin, and phosphorylation of ERK and AKT was evaluated by western blot analysis using phosphospecific and then nonphosphospecific antibodies. Representative results of five independent experiments are shown together with those of densitometric measurement. For the latter, results are mean ± SEM. (e) Chondrocytes were cultured for 7 days with or without echistatin, and cell lysates were obtained. Amounts of active and total RRAS in the lysates were determined by western blot analysis with and without a pull-down assay, respectively. Experiments were repeated five times, and representative bands are shown together with those of densitometric measurement. For the latter, ratios of active RRAS against total RRAS are shown by mean ± SEM. Echistatin was used at 1 μM for these experiments. GADPH, glyceraldehyde 3-phosphate dehydrogenase.